EXTRACELLULAR SECRETION OF TARGET PROTEIN

Abstract

The present invention provides a method for effective extracellular secretion of a target protein, by preparing a fusion protein connecting LARDS to the target protein and having pI lowered by adjusting the overall charge of target protein, and by using ABC transporter of a bacterial type 1 secretion system (T1SS). The method can allows a protein be produced at a large amount simply and effectively without a separate purification process.

Claims

1. An expression vector for extracellular secretion of a target protein in bacteria, comprising an expression cassette including a nucleotide sequence encoding Lipase ABC transporter recognition domain (LARD) and a nucleotide sequence encoding a target protein which are operably linked, wherein the LARD and the target protein have acidic pI and is expressed as a fusion protein.

2. The expression vector of claim 1, wherein the expression vector comprises a nucleotide sequence comprising ABC transporter of bacterial Type 1 Secretion System (T1SS).

3. The expression vector of claim 1, wherein the bacteria comprises an ABC transporter of Type 1 Secretion System (T1SS).

4. The expression vector of claim 2, wherein the ABC transporter of T1SS is a transporter having at least 20% of nucleotide sequence identify with TliDEF transporter of Pseudomonas fluorescence.

5. The expression vector of claim 1, wherein the bacteria is at least one Gram-negative bacteria selected from the group consisting of Pseudomonas sp., Dickeya sp., and Escherichia sp.

6. The expression vector of claim 2, wherein the ABC transporter of T1SS is LipBCD of Serratia marcescens, HasDEF of Serratia marcescens, CyaBDE of Bordetella pertussis, CvaBA+TolC of Escherichia coli, RsaDEF of Caulobacter crescentus, Pseudomonas aeruginosa AprDEF (PaAprDEF), Dickeya dadantii PrtDEF (DdPrtDEF), or Escherichia coli HlyBD+TolC.

7. The expression vector of claim 3, wherein the ABC transporter of T1SS is LipBCD of Serratia marcescens, HasDEF of Serratia marcescens, CyaBDE of Bordetella pertussis, CvaBA+TolC of Escherichia coli, RsaDEF of Caulobacter crescentus, Pseudomonas aeruginosa AprDEF (PaAprDEF), Dickeya dadantii PrtDEF (DdPrtDEF),or Escherichia coli HlyBD+TolCi.

8. The expression vector of claim 1, wherein the nucleotide sequence encoding a target protein further comprises a nucleotide sequence encoding an acidic peptide consisting of 6 to 20 amino acids.

9. The expression vector of claim 1, wherein the target protein is a mutated protein with lowered pI value obtained by deleting at least one of the basic amino acids in the target protein, or by substituting them with other amino acids.

10. The expression vector of claim 9, wherein at least one of the basic amino acids in the target protein is substituted with at least one amino acid selected from the group consisting of acidic amino acids and neutral amino acids.

11. The expression vector of claim 9, wherein the other amino acids is at least one amino acid selected from the group consisting of aspartic acid, glutamic acid, and glutamine.

12. The expression vector of claim 1, wherein the target protein is negatively supercharged protein obtained by performing the following steps: selecting at least an amino acid not affecting the structure of target protein by having a functional group protruding in three dimensional structure of the target protein, and substituting the selected amino acid with at least one selected from the group consisting of acidic amino acids and neutral amino acids, when the selected amino acid is basic.

13. The expression vector of claim 1, wherein the target protein is negatively supercharged protein obtained by performing the following steps: selecting at least an amino acid not affecting the structure of target protein by having a functional group protruding in three dimensional structure of the target protein, mutating at least one selected amino acid, into at least one selected from neutral amino acids and acidic amino acids to produce mutated target protein, and selecting the mutated target protein having activity.

14. A cell comprising an expression vector according to claim 1.

15. A method of performing an extracellular secretion of a target protein in a bacterial cell, comprising: obtaining a target protein with lowered pI by deleting at least one basic amino acid in the target protein, or by substituting them with other amino acids, preparing an expression cassette including a nucleotide sequence encoding Lipase ABC transporter recognition domain (LARD) and a nucleotide sequence encoding a target protein which are operably linked, wherein the LARD and the target protein have acidic pI and is expressed as a fusion protein, and expressing the expression cassette in the bacterial cell.

16. The method of claim 15, wherein the bacterial cell further comprises an ABC transporter of Type 1 Secretion System (T1SS), or an expression cassette comprising a nucleotide sequence encoding ABC transporter of bacterial Type 1 Secretion System (T1SS).

17. The method of claim 15, wherein at least one of the basic amino acids in the target protein is substituted with at least one amino acid selected from the group consisting of acidic amino acids and neutral amino acids.

18. The method of claim 15, wherein the other amino acids is at least one amino acids selected from the group consisting of aspartic acid, glutamic acid, and glutamine.

19. The method of claim 15, wherein the target protein with lowered pI is negatively supercharged protein obtained by performing the following steps: selecting at least an amino acid not affecting the structure of target protein by having a functional group protruding in three dimensional structure of the target protein, and substituting the selected amino acid with at least one selected from the group consisting of acidic amino acids and neutral amino acids, when the selected amino acid is basic.

20. The expression vector of claim 1, wherein the target protein is negatively supercharged protein obtained by performing the following steps: selecting at least an amino acid not affecting the structure of target protein by having a functional group protruding in three dimensional structure of the target protein, mutating at least one selected amino acid, into at least one selected from neutral amino acids and acidic amino acids to produce mutated target protein, and selecting the mutated target protein having activity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0094] FIG. 1a and FIG. 1b confirm the secretion of selected proteins according to Example 6, and represent the western blotting images showing expression and secretion of target proteins.

[0095] FIG. 2a and FIG. 2b show the correlation between the secretion ratio of the target proteins and their isoelectric points according to Example 6. The pI values of the target proteins are calculated for the sequence including attached LARD3.

[0096] FIG. 3a and FIG. 3b are results of expressing Lunasin and derivatives of Lunasin with different length of oligo-aspartic acid tail through LARD3 attachment and confirming secretion, in order to determine the optimal length of the oligo-aspartic acid sequence in P.fluorescens expression and secretion system according to Example 7.

[0097] FIG. 4 shows the structure of the plasmid used in the present invention according to Example 8, and shows the structure of pDART plasmid comprising MCS. Proteins fused with tliD, tliE, tliF and LARD3 are controlled by single operon. In case of (A), as there is the LARD3 gene right behind MCS, the inserted target gene is expressed with LARD3 attached to the C-terminus. (B) shows the structure of pFD10 which is a plasmid in which D10 sequence is attached to the N-terminus. The D10 gene directly follows start codon and is located right before the MCS and LARD3. (C) shows the structure of pBD10 plasmid, which attaches D10 sequence at the C-terminus, but before LARD3. The D10 gene is located between the MCS and LARD3.

[0098] FIG. 5 shows the result of detecting by western blotting and the lipase activity with a measurement medium, after adding 10 aspartic acids (D10) to the N-terminus (FD10) and C-terminus (BD10) of two kinds of TliA lipases (NKC-TliA, CTP-TliA) in which NKC and CTP sequences are attached, respectively, and expressing through pDART plasmid, according to Example 9.

[0099] FIG. 6 shows the result of western blotting detection, after adding 10 aspartic acids (D10) to the N-terminus (FD10) and C-terminus (BD10) of green fluorescent protein (GFP), mannanase, maltose binding protein (MBP) and Thioredoxin, and expressing through pDART plasmid, according to Example 10.

[0100] FIG. 7 shows the result of detecting with western blotting and lipase activity medium, after adding 10 aspartic acids (D10) or 10 arginines (R10), respectively, to the C-terminus of TliA lipase and green fluorescent protein (GFP) according to Example 11.

[0101] FIG. 8 shows the result of adding green fluorescent protein (GFP) supercharged by AvNAPSA method to pDART and expressing it, and detecting it by western blotting, according to Example 12.

[0102] FIG. 9 shows the charge distribution of TliD structure which is ABC protein of TliDEF complex according to Example 5. The parts indicated by circles in A, B, C of FIG. 9 show positively charged parts, and the parts indicated by the circle in D of FIG. 9 show pores inside of the transporter, and the white arrow inside of the circle represents the relatively negative atom and the black arrow represents the relatively positive atom.

[0103] FIG. 10a and FIG. 10b show the comparison of secretion of TliA, CTP-TliA and NKC-TliA according to Example 9, and FIG. 10a is the result of enzyme plate analysis of TliA, CTP-TliA and NKC-TliA, and FIG. 10b shows the western blotting result of TliA, CTP-TliA and NKC-TliA.

[0104] FIG. 11 shows the relation between the protein pI and charge at pH 7.0 according to Example 5.

[0105] FIG. 12 shows the result of predicting the structure of TliD according to Example 5.

[0106] FIG. 13 shows the result of prediction of transmembrane helices of modeled TliD according to Example 5. The rectangular box part corresponds to the transmembrane part predicted by the server.

[0107] FIG. 14 shows the result of ConSurf homologue conservation analysis of modeled TliD according to Example 5. The dark black parts are well conserved parts, and the lighter the color is, the less conserved it is.

[0108] FIG. 15 shows the protein secretion in pDAR-TliA, -NKC (-), NKC-L1, -NKC-L2, NKC-L3, -NKC-TliA according to Example 13. (A) Western blotting of TliA. (B) shows the result of enzyme plate analysis of TliA in different plasmids.

[0109] FIG. 16 shows the result of analysis of secretion of 10SAV, wtSAV, +13SAV and 2-10GST, wtGST, +19GST according to Example 14 (SAV: streptavidin/GST: glutathione 5-transferase).

[0110] FIG. 17 shows the result of inserting and expressing glutathione S-transferase (GST) supercharged by replacing protruding amino acids with aspartic acid or arginine and streptavidin (SAv) to pDART, and detecting with western blotting, while looking at the structure without using AvNAPSA (Average Number of Neighboring Atoms Per Sidechain Atom) method according to Example 14.

[0111] FIG. 18 shows the result of highly negatively charging MelC.sub.2 tyrosinase, cutinase (Cuti), chitinase (Chi) and M37 lipase by AvNAPSA method, and then adding highly negatively charged protein (red) and non-supercharged natural protein corresponding thereto (black) to pDART plasmid, respectively, and expressing them, and detecting by western blotting, according to Example 15.

[0112] FIG. 19 shows the experimental result of measuring the degree of protein secretion in the enzyme activity measurement medium through the color change of the colony peripheral medium, after simultaneously expressing TliA protein (original substrate of TliDEF transporter) and T1SS transporters isolated from different 3 kinds of bacteria, according to Example 16.

[0113] FIG. 20 shows the experimental result of measuring the degree of protein secretion in the enzyme activity measurement medium through the color change of the colony peripheral medium, by suspending the LARD3 signal sequence to cutinase protein (Cuti) and highly negatively charged cutinase protein (Cuti(-)) and then expressing them together with different 3 kinds of T1SS transporter proteins in E. coli according to Example 17.

[0114] FIG. 21 shows the experimental result of detecting the protein concentration inside and outside the cell by western blotting, after attaching the LARD3 signal sequence to cutinase protein (Cuti) and highly negatively charged cutinase protein (Cuti(-)) and then expressing them together with different 3 kinds of T1SS transporter proteins in E. coli and liquid culturing them, according to Example 18.

[0115] FIG. 22 shows the experimental result of detecting the protein concentration inside and outside of the cell by western blotting, after attaching LARD3 signal sequence to M37 lipase protein (M37) and highly negatively charged M37 lipase protein (M37(-)), and then expressing them with different 3 kinds of T1SS transporter proteins in E. coli by performing liquid culture according to Example 19.

[0116] FIG. 23 shows the sequence identity between TliDET transporter and various T1SS transporters and the proportion of the portion of similar sequence in the full sequence.

[0117] FIG. 24 shows the amino acid sequences of wild type M37 and mutants with modified amino acids by using the selective superneutralization of the positive charges and the random mutagenesis-screening method.

[0118] FIG. 25 shows the experimental result of detecting the protein concentration inside and outside the cell by western blotting, measuring the degree of protein secretion in the enzyme activity measurement medium through the color change of the colony peripheral medium, and the mixed-based codon strategy utilized to prepare M37(var) mutant according to Examples 21 and 22.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0119] Hereinafter, the present invention will be described in detail by examples. However, the following examples are intended to illustrate the present invention only, but the present invention is not limited by the following examples.

[Example 1] Bacterial Strains and Growth Media

[0120] Plasmid construction and gene cloning were performed in E. coli XL1-BLUE. Protein expression and secretion were observed in the P. fluorescens tliA prtA strain, which is a double-deletion derivative of P. fluorescens SIK-W 1 (Son, M., Moon, Y., Oh, M. J., Han, S. B., Park, K. H., Kim, J G., and Ahn, J. H. (2012) Lipase and protease double-deletion mutant of Pseudomonas fluorescens suitable for extracellular protein production. Appl Environ Microbiol 78, 8454-8462). Microorganisms were cultured in lysogeny broth (LB) with 30 g/ml kanamycin. An enzyme plate assay for the target genes with lipase activity (TliA, NKC-TliA, and CTP-TliA) was prepared with LB agar media containing blender-mixed 0.5% colloidal glyceryl tributyrate. E. coli and P. fluorescens were incubated at 37 C. and 25 C., respectively. E. coli transformation was performed following the standard heat-shock method, and P. fluorescens transformation was performed via electroporation at 2.5 kV, 125, and 50 F, with electrocompetent cells prepared using a standard electroporation protocol (Ausubel, M. F. (2014) Escherichia coli, Plasmids, and Bacteriphages. in Current Protocols in Molecular Biology, John Wiley & Sons, Inc. pp). The transformed P. fluorescens were cultured in test tubes with 5 ml of liquid LB media, including 60 g/ml kanamycin, and were incubated at 25 C. in a 180 rpm shaking incubator until the stationary phase was reached. The proteins were analyzed for both expression and secretion by seeding the transformed cells in liquid LB or streaking them on the solid-plate activity assay.

[Example 2] Plasmid Vector Constructions

[0121] Plasmid pDART was used for the secretory production of different proteins of the present inventors (Ryu, J., Lee, U., Park, J., Yoo, D. H., and Ahn, J. H. (2015) A vector system for ABC transporter-mediated secretion and purification of recombinant proteins in Pseudomonas species. Appl Environ Microbiol 81, 1744-1753). Plasmid vectors pFD10 and pBD10 were derivatives of pDART, constructed by adding codons for 10 aspartic acid residues to the target proteins in either the upstream or downstream position of MCS. The DNA sequence for 10 aspartic acids was amplified via PCR using synthesized Glycine max lunasin gene (Galvez, A. F., Chen, N., Macasieb, J., and de Lumen, B. O. (2001) Chemopreventive Property of a Soybean Peptide (Lunasin) That Binds to Deacetylated Histones and Inhibits Acetylation. Cancer Research 61, 7473-7478) as a template. Two different PCR products were obtained, each for pFD10 and pBD10. One or two arbitrary bases are inserted upstream or downstream of the primers to keep the translation in-frame, causing a slight size and pI difference between the pFD10- and pBD10-inserted proteins.

[0122] Then, recombining the PCR product with pDART to construct pFD10 and pBD10 was accomplished with an In-Fusion cloning kit (Clontech In-Fusion HD cloning plus CE). To linearize pDART, it was digested with either XbaI (pFD10 construction) or SasI (pBD10). Then, the linearized pDART and the corresponding PCR products were digested with In-Fusion 3-to-5-exodeoxyribonuclease and re-ligated following the standard protocol of the In-Fusion kit. Ligation of these DNA fragments with complementary 15-base 5-overhangs resulted in pFD10 and pBD10 plasmid, ready for target gene insertions. pDART, pFD10, and pBD10 sequences near their MCSs are provided in Table 2.

[0123] The amino acid sequences underlined in the following Table 2 represent LARD3 signal sequences, and the bold IEGR is a residue that connects the target protein and LARD3 signal sequence, and is a part that Factor Xa recognizes and cleaves.

[0124] The target protein may be further purified from Factor Xa and LARD3 by purification tag such as His-tag.

[0125] The description of each part of the sequence of the following Table 2 was disclosed in FIG. 19a to FIG. 19g. FIG. 19a to FIG. 19f represent the total sequence of target proteins in FASTA format, and FIG. 19g represents color codes for indicating enzyme sites and polypeptide characteristics.

TABLE-US-00002 TABLE2 Fullsequencesofthetargetproteins,inFASTAformats SEQIDNO TliA,wildtype MGVFDYKNLGTEASKTLFADATAITLYTYHNLDNGFAVGYQQHGLGLGLPATLVGALLG 1 (asareference) STDSQGVIPGIPWNPDSEKAALDAVHAAGWTPISASALGYGGKVDARGTFFGEKAGYTT AQAEVLGKYDDAGKLLEIGIGFRGTSGPRESLITDSIGDLVSDLLAALGPKDYAKNYAG EAFGGLLKTVADYAGAHGLSGKDVLVSGHSLGGLAVNSMADLSTSKWAGFYKDANYLAY ASPTQSAGDKVLNIGYENDPVFRALDGSTFNLSSLGVHDKAHESTTDNIVSFNDHYAST LWNVLPFSIANLSTWVSHLPSAYGDGMTRVLESGFYEQMTRDSTIIVANLSDPARANTW VQDLNRNAEPHTGNTFIIGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHF GQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEG VLIS TliA,expressed MSRMGVFDYKNLGTEASKTLFADATAITLYTYHNLDNGFAVGYQQHGLGLGLPATLVGA 2 inpDARTplasmid LLGSTDSQGVIPGIPWNPDSEKAALDAVHAAGWTPISASALGYGGKVDARGTFFGEKAG (thisisusedfor YTTAQAEVLGKYDDAGKLLEIGIGFRGTSGPRESLITDSIGDLVSDLLAALGPKDYAKN computational YAGEAFGGLLKTVADYAGAHGLSGKDVLVSGHSLGGLAVNSMADLSTSKWAGFYKDANY analysis) LAYASPTQSAGDKVLNIGYENDPVFRALDGSTFNLSSLGVHDKAHESTTDNIVSFNDHY ASTLWNVLPFSIANLSTWVSHLPSAYGDGMTRVLESGFYEQMTRDSTIIVANLSDPARA NTWVQDLNRNAEPHTGNTFIIGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFS GHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLW SEGVLISELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRI IGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS NKC-TliA:NKC MSRHMGTAPKAMKLLKKLLKLQKKGIGSMGVFDYKNLGTEASKTLFADATAITLYTYHN 3 ismarkedcyan LDNGFAVGYQQHGLGLGLPATLVGALLGSTDSQGVIPGIPWNPDSEKAALDAVHAAGWT PISASALGYGGKVDARGTFFGEKAGYTTAQAEVLGKYDDAGKLLEIGIGFRGTSGPRES LITDSIGDLVSDLLAALGPKDYAKNYAGEAFGGLLKTVADYAGAHGLSGKDVLVSGHSL GGLAVNSMADLSTSKWAGFYKDANYLAYASPTQSAGDKVLNIGYENDPVFRALDGSTFN LSSLGVHDKAHESTTDNIVSFNDHYASTLWNVLPFSIANLSTWVSHLPSAYGDGMTRVL ESGFYEQMTRDSTIIVANLSDPARANTWVQDLNRNAEPHTGNTFIIGSDGNDLIQGGKG ADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKA VGADTVLSFGADSVTLVGVGLGGLWSEGVLISELIEGRGSDGNDLIQGGKGADFIEGGK GNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLS FGADSVTLVGVGLGGLWSEGVLIS CTP-TliA:CTP MSRMRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSMYGRRARRRRRRSMAGTGGM 4 ismarkedcyan GVFDYKNLGTEASKTLFADATAITLYTYHNLDNGFAVGYQQHGLGLGLPATLVGALLGS TDSQGVIPGIPWNPDSEKAALDAVHAAGWTPISASALGYGGKVDARGTFFGEKAGYTTA QAEVLGKYDDAGKLLEIGIGFRGTSGPRESLITDSIGDLVSDLLAALGPKDYAKNYAGE AFGGLLKTVADYAGAHGLSGKDVLVSGHSLGGLAVNSMADLSTSKWAGFYKDANYLAYA SPTQSAGDKVLNIGYENDPVFRALDGSTFNLSSLGVHDKAHESTTDNIVSFNDHYASTL WNVLPFSIANLSTWVSHLPSAYGDGMTRVLESGFYEQMTRDSTIIVANLSDPARANTWV QDLNRNAEPHTGNTFIIGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFG QDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGV LISELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQ PTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS Mannanase MSRHHHHHHTVSPVNPNAQQTTKAVMNWLAHLPNRTENRVLSGAFGGYSHDTFSMAEAD 5 (Mann) RIRSATGQSPAIYGCDYARGWLETANIEDSIDVSCNSDLMSYWKNDGIPQISLHLANPA FQSGHFKTPITNDQYKKILDSSTAEGKRLNTMLSKIADGLQELENQGVPVLFRPLHEMN GERFWWGLTSYNQKDNERISLYKQLYKKIYHYMTDTRGLDHLIWVYSPDANRDFKTDFY PGASYVDIVGLDAYFQDAYSINGYDQLTALNKPFAFTEVGPQTANGSFDYSLFINAIKH RYPKTIYFLAWNDEWSPAVNKGASALYHDSWTLNKGEIWNGDSLTPIVEELIEGRGSDG NDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGS TDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS Musseladhesion MSSMRGSHHHHHHGSASAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKGCSSEEYKGGYY 6 protein(MAP): PGNSNHYHSGGSYHGSGYHGGYKGKYYGKAKKYYYKYKNSGKYKYLKKARKYHRKGYKK usedSpeI-SacI YYGGSSEFAKPSYPPTYKAKPSYPPTYKAKPSYPPTYKELIEGRGSDGNDLIQGGKGAD insertion FIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVG ADTVLSFGADSVTLVGVGLGGLWSEGVLIS Maltosebinding MSRKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDG 7 protein(MBP) PDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSL IYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENG KYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWS NIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEA VNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAA SGRQTVDEALKDAQTRITKELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNT FLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGL GGLWSEGVLIS Thioredoxin MSRMLHQQRNQHARLIPVELYMSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKM 8 (Trx) IAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSK GQLKEFLDANLAELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHF GQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEG VLIS Cutinase(Cuti) MSRHHHHHHAPTSNPAQELEARQLGRTTRDDLINGNSASCADVIFIYARGSTETGNLGT 9 LGPSIASNLESAFGKDGVWIQGVGGAYRATLGDNALPRGTSSAAIREMLGLFQQANTKC PDATLIAGGYSQGAALAAASIEDLDSAIRDKIAGTVLFGYTKNLQNRGRIPNYPADRTK VFCNTGDLVCTGSLIVAAPHLAYGPDARGPAPEFLIEKVRAVRGSALEELIEGRGSDGN DLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGST DLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS Chitinase(Chi) MSRHHHHHHANSPKQSQKIVGYFPSWGVYGRNYQVADIDASKLTHLNYAFADICWNGKH 10 GNPSTHPDNPNKQTWNCKESGVPLQNKEVPNGTLVLGEPWADVTKSYPGSGTTWEDCDK YARCGNFGELKRLKAKYPHLKTIISVGGWTWSNRFSDMAADEKTRKVFAESTVAFLRAY GFDGVDLDWEYPGVETIPGGSYRPEDKQNFTLLLQDVRNALNKAGAEDGKQYLLTIASG ASRRYADHTELKKISQILDWINIMTYDFHGGWEATSNHNAALYKDPNDPAANTNFYVDG AINVYTNEGVPVDKLVLGVPFYGRGWKSCGKENNGQYQPCKPGSDGKLASKGTWDDYST GDTGVYDYGDLAANYVNKNGFVRYWNDTAKVPYLYNATTGTFISYDDNESMKYKTDSIK TKGLSGAMFWELSGDCRTSPKYSCSGPKLLDTLVKELLGGPINQKDTEPPTNVKNIVVT NKNSNSVQLNWTASTDNVGVTEYEITAGEEKWSTTTNSITIKNLKPNTEYKFSIIAKDA AGNKSQPTALTVKTDEANMTPPDGNGTATFSVTSNWGSGYNFSIIIKNNGTNPIKNWKL EFDYSGNLTQVWDSKISSKTNNHYVITNAGWNGEIPPGGSITIGGAGTGNPAELLNAVI SENELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQ PTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS M37lipase MSRHMSYTKEQLMLAFSYMSYYGITHTGSAKKNAELILKKMKEALKTWKPFQEDDWEVV 11 (M37) WGPAVYTMPFTIFNDAMMYVIQKKGAEGEYVIAIRGTNPVSISDWLFNDFMVSAMKKWP YASVEGRILKISESTSYGLKTLQKLKPKSHIPGENKTILQFLNEKIGPEGKAKICVTGH SKGGALSSTLALWLKDIQGVKLSQNIDISTIPFAGPTAGNADFADYFDDCLGDQCTRIA NSLDIVPYAWNTNSLKKLKSIYISEQASVKPLLYQRALIRAMIAETKGKKYKQIKAETP PLEGNINPILIEYLVQAAYQHVVGYPELMGMMDDIPLTDIFEDAIAGLLLEHHHHHHGT ASELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQP TDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS Capsid(Cap), MSRMARKKSTPRRRKAVKRRRTVRRRQSPKARVRSTTTKAKRRISPSGSGSQHLTVRKQ 12 Chaetoceros PFSNATSQPKILDGALTSSLSRRLQNVIGLTNGNGGLGTEIMHIFFAPTLGIPLIAMNS salsugineum AEGVALRPSSSADPFFIGFPGQTIKFDYVSSGTTPPATGNLVTFSNECGFSKWRIVSQG nuclearinclusion LRMELANSDEENDGWFEAVRFNWRNNPADICFTPIDGTLGGAKTTDFAVAPSPVGMYAL virus(CsNIV KDMAMVEQPGYTTGLLKDLKNHEFMLHPQSTTHDPIILEQSYEGTIGTTAADDMYYSVT SEVFELGNTVRGNTMKNSLVDNNMDWIYLRLHCRTNNGTTSNGSKLIVNAIQNLEVSFN PSSDFAAFQTINKMHPQQKKVDDQLNNSAEASNKRQKTGGGELIEGRGSDGNDLIQGGK GADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAK AVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS DnaJ(Hsp40) MSRMAKQDYYEILGVSKTAEEREIRKAYKRLAMKYHPDRNQGDKEAEAKFKEIKEAYEV 13 LTDSQKRAAYDQYGHAAFEQGGMGGGGFGGGADFSDIFGDVFGDIFGGGRGRQRAARGA DLRYNMELTLEEAVRGVTKEIRIPTLEECDVCHGSGAKPGTQPQTCPTCHGSGQVQMRQ GFFAVQQTCPHCQGRGTLIKDPCNKCHGHGRVERSKTLSVKIPAGVDTGDRIRLAGEGE AGEHGAPAGDLYVQVQVKQHPIFEREGNNLYCEVPINFAMAALGGEIEVPTLDGRVKLK VPGETQTGKLFRMRGKGVKSVRGGAQGDLLCRVVVETPVGLNERQKQLLQELQESFGGP TGEHNSPRSKSFFDGVKKFFDDLTRGTASELIEGRGSDGNDLIQGGKGADFIEGGKGND TIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGA DSVTLVGVGLGGLWSEGVLIS Endo-1,4-- MSRHHHHHHYKATTTRYYDGQEGACGCGSSSGAFPWQLGIGNGVYTAAGSQALFDTAGA 14 glucanaseV SWCGAGCGKCYQLTSTGQAPCSSCGTGGAAGQSIIVMVTNLCPNNGNAQWCPVVGGTNQ (Eg15) YGYSYHFDIMAQNEIFGDNVVVDFEPIACPGQAASDWGTCLCVGQQETDPTPVLGNDTG STPPGSSPPATSSSPPSGGGQQTLYGQCGGAGWTGPTTCQAPGTCKVQNQWYSQCLPGT ASELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQP TDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS Green MSRMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVP 15 fluroescent WPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGNYKTRAEVKFE protein(GFP) GDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIKANFKIRHNIEDG SVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGM DELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPT DRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS 30Negatively MSRMGHHHHHHGGASKGEELFDGVVPILVELDGDVNGHEFSVRGEGEGDATEGELTLKF 16 supercharged ICTTGELPVPWPTLVTTLTYGVQCFSDYPDHMDQHDFFKSAMPEGYVQERTISFKDDGT GFP(GFP-(-30)) YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHDVYITADKQENGIKAE FEIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDDHYLSTESALSKDPNEDRDHMVLLEF VTAAGIDHGMDELYKELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFS GHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLW SEGVLIS +36Positively MSRMGHHHHHHGGASKGERLFRGKVPILVELKGDVNGHKFSVRGKGKGDATRGKLTLKF 17 supercharged ICTTGKLPVPWPTLVTTLTYGVQCFSRYPKHMKRHDFFKSAMPKGYVQERTISFKKDGK GFP(GFP-(+36)) YKTRAEVKFEGRTLVNRIKLKGRDFKEKGNILGHKLRYNFNSHKVYITADKRKNGIKAK FKIRHNVKDGSVQLADHYQQNTPIGRGPVLLPRNHYLSTRSKLSKDPKEKRDHMVLLEF VTAAGIKHGRDERYKELIEGRGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFS GHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLW SEGVLIS Epidermal MNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELRSRIEG 18 growthfactor RGSDGNDLIQGGKGADFIEGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQ (EGF) GADGSTDLRDHAKAVGADTVLSFGADSVTLVGVGLGGLWSEGVLIS Alkaline MSSMPVLENRAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEIT 19 phosphatase(AP) AARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTDSAASATAWSTGVKTYNG ALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQDATPAALVAHVTSRKCYGPSATSEK CPGNALEKGGKGSITEQLLNARADVTLGGGAKTFAETATAGEWQGKTLREQAQARGYQL VSDAASLNSVTEANQQKPLLGLFADGNMPVRWLGPKATYHGNIDKPAVTCTPNPQRNDS VPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQDHAANPCGQIGETVDLDEAVQRALE FAKKEGNTLVIVTADHAHASQIVAPDTKAPGLTQALNTKDGAVMVMSYGNSEEDSQEHT GSQLRIAAYGPHAANVVGLTDQTDLFYTMKAALGLKELIEGRGSDGNDLIQGGKGADFI EGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGAD TVLSFGADSVTLVGVGLGGLWSEGVLIS Phospholipase MSMSLSFTSAIAPAAIQPPMVRTQPEPLSSSQPVEASATKAPVATLSQNSLNAQSLLNT 20 A1(PLA1) LVSEISAAAPAAANQGVTRGQQPQKGDYTLALLAKDVYSTGSQGVEGFNRLSADALLGA GIDPASLQDAASGFQAGIYTDNQQYVLAFAGTNDMRDWLSNVRQATGYDDVQYNQAVSL AKSAKAAFGDALVIAGHSLGGGLAATAALATGTVAVTFNAAGVSDYTLNRMGIDPAAAK QDAQAGGIRRYSEQYDMLTGTQESTSLIPDAIGHKITLANNDTLSGIDDWRPSKHLDRS LTAHGIDKVISSMAEQKPWEAMANAHHHHHHGTASELIEGRGSDGNDLIQGGKGADFIE GGKGNDTIRDNSGHNTFLFSGHFGQDRIIGYQPTDRLVFQGADGSTDLRDHAKAVGADT VLSFGADSVTLVGVGLGGLWSEGVLIS

TABLE-US-00003 TABLE3 FactorXa IEGR 21 LARD3signal GSDGNDLIQGGKGADFIEGGKGNDTIRDNSGH 22 peptide NTFLFSGHFGQDRIIGYQPTDRLVFQGADGST DLRDHAKAVGADTVLSFGADSVTLVGVGLGGL WSEGVLIS pDART MSRHMGTASELIEGRGSDGNDLIQGGKGADFI 23 Translation EGGKGNDTIRDNSGHNTFLFSGHFGQDRIIGY Structure QPTDRLVFQGADGSTDLRDHAKAVGADTVLSF GADSVTLVGVGLGGLWSEGVLIS pFD10 MSSDDDDDDDDDDSRHMGTASELIEGRGSDGN 24 Translation DLIQGGKGADFIEGGKGNDTIRDNSGHNTFLF Structure SGHFGQDRIIGYQPTDRLVFQGADGSTDLRDH AKAVGADTVLSFGADSVTLVGVGLGGLWSEGV LIS pBD10 MSRHMGTASELDDDDDDDDDDDIEGRGSDGND 25 Translation LIQGGKGADFIEGGKGNDTIRDNSGHNTFLFS Structure GHFGQDRIIGYQPTDRLVFQGADGSTDLRDHA KAVGADTVLSFGADSVTLVGVGLGGLWSEGVL IS pBE10 MSRHMGTASELEEEEEEEEEEGIEGRGSDGND 26 Translation LIQGGKGADFIEGGKGNDTIRDNSGHNTFLFS Structure GHFGQDRIIGYQPTDRLVFQGADGSTDLRDHA KAVGADTVLSFGADSVTLVGVGLGGLWSEGVL IS pBH10 MSRHMGTASELHHHHHHHHHHGIEGRGSDGND 27 Translation LIQGGKGADFIEGGKGNDTIRDNSGHNTFLFS Structure GHFGQDRIIGYQPTDRLVFQGADGSTDLRDHA KAVGADTVLSFGADSVTLVGVGLGGLWSEGVL IS pBR10 MSRHMGTASELRRRRRRRRRRGIEGRGSDGND 28 Translation LIQGGKGADFIEGGKGNDTIRDNSGHNTFLFS Structure GHFGQDRIIGYQPTDRLVFQGADGSTDLRDHA KAVGADTVLSFGADSVTLVGVGLGGLWSEGVL IS Color code for enzyme sites and polypeptide features >Multiple cloning site (MCS): XbaI: tctaga, SR NdeI: catatg, HM KpnI: ggtacc, GT NheI: gctagc, AS SacI: gagctc, E:

[Example 3] Construction of Plasmids with Inserted Target Genes

[0126] Thirteen target genes were selected for pDART insertion. The genes were amplified with PCR from extracted genomic DNA samples (TliA, MBP, Trx, and Hsp40), total cDNA (Eg1V), synthesized DNA products (NKC-TliA, CTP-TliA, MAP, lunasin, lunasin derivatives, GFP, and supercharged GFPs), or plasmids (other proteins), or the like.

[0127] Their N-terminal signal peptides were detected with the SignalP 4.1 web-based prediction algorithm (http://www.cbs.dtu.dk/services/SignalP/) (Petersen, T. N., Brunak, S., von Heijne, G., and Nielsen, H. (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nature methods 8, 785-786) and were excluded from cloning and expression processes. For synthetic genes, the codons were optimized for either E. coli expression (supercharged GFPs) or P. fluorescens expression (TliA derivatives).

[0128] The lunasin gene was synthesized and amplified with PCR for pDART insertion. With various primers, we also synthesized its variations with differing lengths of Asp polypeptide tail at their C terminus such as lunasin-DO, lunasin-D5, lunasin-D15, and lunasin-D20 (FIG. 3B). NKC-TliA and CTP-TliA are derivatives of TliA. NKC is an antibiotic polypeptide developed previously, and CTP is a cytoplasmic transduction peptide that was developed as a cellular import tag previously. We have synthesized genes for these two, with codons optimized for P. fluorescens expression.

[0129] The supercharged variations of GFP, including negatively supercharged GFP (-30) and positively supercharged GFP (+36), were previously developed by replacing solvent-exposed residues of GFP with negatively or positively charged amino acids. We have completely synthesized genes that code for these two supercharged proteins, with codons optimized for E. coli expression.

[0130] The primers we used for PCR had restriction enzyme sites that were utilized to insert the target genes to the MCSs of the plasmids (pDART, pFD10, and pBD10). The PCR products and plasmid vectors were double-digested with two restriction enzymes for XbaI, KpnI, SacI, or SpeI (which is compatible with XbaI). The specific pair of enzymes used on each gene can be directly identified from the full sequences provided in Table 2.

[0131] Then, the plasmid treated by restriction enzyme and the gene were ligated with T4 ligase. The constructed plasmids were then introduced into E. coli for cloning, and the cloned plasmids were first obtained using a standard plasmid purification method. The purified plasmids were then introduced to P. fluorescens, for which expression and secretion were analyzed.

[Example 4] Western Blotting Conditions

[0132] After 48 h of cell growth (secretion occurs during the entire growth), the liquid culture reached stationary growth phase, and the cell density reached about 1.510.sup.9 cells/ml (OD600=3). Then, 400 l of the liquid cultures were taken and centrifuged at 18,000 rcf for 10 min to separate the supernatant and the cell pellet. 16 l of culture (0.048 OD) equivalents of the cell pellet extract and supernatant were each loaded onto 10% polyacrylamide gels. SDS-PAGE was used to separate the proteins according to their sizes.

[0133] Then, the proteins were transferred to a nitrocellulose membrane (Amersham) for Western blotting. Polyclonal anti-LARDS rabbit immunoglobulin G (IgG) and anti-neomycin phosphotransferase 2 (Abcam, ab33595) were utilized as the primary antibody with 1:3000 and 1:500 dilution each, and anti-rabbit recombinant goat IgG-peroxidase (anti-rIgG goat IgG-peroxidase) was used as the secondary antibody with 1:1000 dilution. The bands were then detected using a chemiluminescence agent (Advansta WesternBright Pico). Western blotting images were acquired using an Azure C600 automatic detecting system. All included Western blotting images are representative results from at least three different repeated experiments, starting over again from cell culturing with independent P. fluorescens colonies.

[0134] After the images were obtained, the results of experiment 1 (FIG. 1) was quantified with ImageJ software. Then, % secretion of the target proteins of this experiment was calculated. The % secretion was calculated as follows.

% secretion=S.sub.supernatant/(S.sub.supernatant+S.sub.cell)100%

[0135] where S is the normalized signal strength of each bands in the Western blotting image, and the subscripts denote the sample type of the lanes.

[Example 5] Analysis of Polypeptide Properties and Protein Structure

[0136] The theoretical pI values of the target proteins were calculated using the ExPASy Compute pI/Mw tool (Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J. C., Williams, K. L., Appel, R. D., and Hochstrasser, D. F. (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112, 531-552). The entire sequences were used, and LARD3 and any additional sequences from the enzyme sites were included in the sequences for this purpose. The protein pI values are highly correlated with their charge per residue, and the correlation analysis of the protein pI values and their charge per residue is included in FIG. 11.

[0137] FIG. 11 shows relationship between protein pI and their charges at pH 7.0. Isoelectric points and charge per 100 residues of the LARD3-attached recombinant proteins show highly linear correlation. Wild-type TliA is marked in blue. Proteins that were observed not to be secreted to the extracellular culture were marked in red. As a result, a clear linear correlation is observed. The estimated unfolded protein charge at pH 7.0 is calculated by Protein Calculator v3.4 (http://protcalc.sourceforge.net/cgi-bin/protcalc).

[0138] Then, SWISS-MODEL structural homology modeling (https://swissmodel.expasy.org/) was used to study the ABC transporter protein structures (Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22, 195-201).

[0139] The present inventors used A. aeolicus PrtD (PDB code 5122) (Morgan, J. L. W., Acheson, J. F., and Zimmer, J. (2017) Structure of a Type-1 Secretion System ABC Transporter. Structure 25, 522-529) as a template, with sequence identity of 40.98%. The result of prediction of the structure of TliD was shown in FIG. 12.

[0140] FIG. 12 shows the structure prediction result of TliD and alignment with template, colored according to QMEAN4 score. Residues with low prediction degree (light color part of FIG. 12) are mainly located on the external surface, typically on random coils and protrusion parts. QMEAN4 score and coloring were obtained by SWISS-MODEL.

[0141] The model's transmembrane helices were verified by DAS-TMfilter (http://mendel.imp.ac.at/sat/DAS/) (Cserzo, M., Eisenhaber, F., Eisenhaber, B., and Simon, I. (2002) On filtering false positive transmembrane protein predictions. Protein Engineering, Design and Selection 15, 745-752), and the results are provided in FIG. 13.

[0142] FIG. 13 shows transmembrane helices prediction result of modeled TliD. The prediction was obtained by DAS-TMfilter webserver. (A) Predicted structure of TliD dimer, with transmembrane helices marked with different colors. (B) Sequence of TliD, highlighted with the identical color-codes with (A).

[0143] The surface of the obtained 3D model was calculated with Swiss PdbViewer (spdbv) (http://spdbv.vital-it.ch/) and colored according to the charge. The present inventors used the ConSurf web server (http://consurf.tau.ac.il/2016/) to compare TliD with its homologs and to verify the structure prediction of TliD (Ashkenazy, H., Abadi, S., Martz, E., Chay, O., Mayrose, I., Pupko, T., and Ben-Tal, N. (2016) ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Research 44, W344-W350). FIG. 14 includes information of conserved residues of TliD.

[0144] FIG. 14 shows the Consurf homologue conservation analysis result of modeled TliD. The present inventors ran a ConSurf homolog conservation analysis on TliD. Multiple Sequence Alignment were built using MAFFT, the homologues were collected from UniProt, homologue search algorithm BLAST, PSI-BLAST E-value 0.001, number of PSI-BLAST Iterations 5, % ID Between Sequences 25-95%. 197 unique proteins were scanned, and among them, 50 sequences closest to the query were used. Phylogenic neighbors were scanned with ML distance, and the conservation score was calculated with Bayesian algorithm. (A) TliD dimer, colored according to the Bayesian conservation score. The transmembrane helices of TliD were conserved among the homologues. Specifically, the residues facing inside of the TliD's central channel were highly conserved, while residues facing outside of the central channel (facing the phospholipids or the cytoplasm) were highly variable.

[0145] The ConSurf homology analysis also approved our structural prediction in a sense that most of the transmembrane helices were highly conserved in the inner surface-facing residues, and this makes our structural prediction even more persuasive. Finally, the present inventors checked side-chain pKa values of the highly conserved arginine and lysine residues at the potentially important positions (C, D and F of FIG. 9) with the web-based PDB 2PQR server (http://nbcr-222.ucsd.edu/pdb2pqr_2.0.0/).

[0146] In the subsequently progressed homology-based structure prediction model, it was shown that the dimer of this protein has positive charge distribution at the inner surface of the channel (FIG. 9A and FIG. 9B). This prediction model was prepared using Aquifex aeolicus PrtD (PDB code 5122) with sequence identity of 40.98% as a template. Moreover, a ConSurf homolog conservation analysis on TliD showed that these charges were indeed conserved, forming a positively charged sub-region at the midpoint of the channel (FIG. 9C and FIG. 9D). In addition, on the kinked helix on the substrate entry window, there is a positively-charged residue that sticks out toward the pore of the window and blocks the window in ADP-bound state of TliD. The ConSurf results also verified that this residue was charge-conserved, as all of the 50 homologs had either arginine or lysine at this residue (FIG. 9C, black arrow). The present inventors expect that this positively-charged inner surface interacts with negatively-charged residues during protein transport, facilitating secretion (FIG. 9E).

[0147] FIG. 9 shows the charge distribution in the structure of TliD, the ABC protein of the TliDEF complex. (A) electron repulsion surface of the TliD monomer. Colored according to its surface electric potential, from blue (+7 kBT/e) to red (-7 kBT/e). The inner surface of the central channel with highly positive charge is circled on top. (B) TliD homodimer, with one of the monomers presented in the ribbon model. The inner surface of the central channel with positive charge is circled on top and substrate entry window is circled on bottom. (C) TliD. The conserved positive charge cluster at the midpoint of the channel's inner surface are circled. The two -helices that form substrate entry window are ovaled. Among the two conserved positively-charged residues, Arg-316 (black arrow) sticks out to the pore. (D) TliD dimer, seen from the periplasmic face. Positive charges are located in the middle of the channel (circled yellow), whereas negative charges are outside of the channel (E) schematic model of the TliD dimer, transporting a highly negatively charged recombinant polypeptide with the attached LARD3. The NBD (nucleotide-binding domain) and transmembrane domain (TMD) of TliD are labeled accordingly. The electric potential across the inner membrane (IM) is 150 mV, where the cytoplasm (CP) is more negative than the periplasm (PP). This potential difference also makes it more favorable to outward-transport negatively-charged proteins than positively-charged proteins.

[0148] The present inventors visualized the results with the PyMOL software. All sequences that were used for the analysis are provided in Table 2 and Table 3.

[Example 6] Cross-Analyzing the Secretion of Recombinant Proteins and their pI

[0149] Thirteen genes (Table 4) of target proteins of different sizes, flexibility, volume, weight, etc. were introduced to P. fluorescens tliA prtA via pDART, where they are attached to a C-terminal LARD3 signal sequence. Table 4 represents the list of genes and their references, and genes indicated by * represent genes which were not used in the present experiment, but were secreted in the previous research. Then, after liquid culturing the cells, the supernatant and cell pellet were analyzed via Western blotting (FIG. 1a and FIG. 1b).

TABLE-US-00004 TABLE 4 Code Protein name Source DNA type TliA Thermostable Pseudomonas fluorescens SIK-W1 Genomic lipase A DNA NKC- NKC-TliA Yang, K. S., Sung, B. H., Park, M. K., Synthesized TliA Lee, J. H., Lim, K. J., Park, S. C., Kim, S. J., Kim, H. K., Sohn, J. -H., Kim, H. M., and Kim, S. C. (2015) Recombinant Lipase Engineered with Amphipathic and Coiled-Coil Peptides. ACS Catalysis 5, 5016-5025 CTP- CTP-TliA Kim, D., Jeon, C., Kim, J. -H., Kim, Synthesized TliA M. -S., Yoon, C. -H., Choi, I. -S., Kim, S. -H., and Bae, Y. -S. (2006) Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo. Experimental Cell Research 312, 1277-1288 Mann Mannanase Bacillus subtilis Plasmid MAP Mussel adhesion MAP fp-151 Synthesized protein MBP Maltose binding Escherichia coli XL1-Blue Genomic protein DNA Trx Thioredoxin Escherichia coli XL1-Blue Genomic DNA Cuti Cutinase Nectria haematococca Plasmid Chi Chitinase Bacillus thuringenesis Plasmid M37 M37 lipase Photobacterium lipolyticum Plasmid Cap Capsid protein Chaetoceros salsugineum Plasmid DNA inclusion virus Hsp40 DnaJ charperone Escherichia coli XL1-Blue Genomic DNA EglV Endo-1,4-- Trichoderma reesei QM6a Total glucanase V cDNA GFP Green fluorescent pGFPuv (Clontech) Plasmid protein GFP(30) Negatively Lawrence, M. S., Phillips, K. J., and Synthesized supercharged GFP Liu, D. R. (2007) Supercharging proteins can impart unusual resilience. Journal of the American Chemical Society 129, 10110-10112 GFP(+36) Positively Lawrence, M. S., Phillips, K. J., and Synthesized supercharged GFP Liu, D. R. (2007) Supercharging proteins can impart unusual resilience. Journal of the American Chemical Society 129, 10110-10112 EGF Epidermal growth Homo sapiens Plasmid factor AP Alkaline phosphatase Escherichia coli XL1-Blue Genomic DNA PLA1 Phospholipase A.sub.1 Serracia marescens Plasmid

[0150] As shown in FIG. 1a and FIG. 1b, Mannanase, MBP, NKC-TliA, Eg1V, GFP, and thioredoxin were both detectable in the cell pellet and the supernatant, showing successful expression and secretion out to the extracellular media. However, MAP, cutinase, chitinase, capsid, Hsp40, and CTP-TliA were not detected in the supernatant despite being detected in the cell pellet, signifying that they were not secreted.

[0151] FIG. 1a and FIG. 1b confirm secretion of selected proteins, and represent western blotting image showing the expression and secretion of the target proteins. The cell samples show the amount of the protein that remains in the cytoplasm, and the supernatant samples represent the amount of protein that is localized to the extracellular space. For comparison, equivalent amounts of cell extract and culture supernatant (16 l) were loaded onto the gel and were analyzed via Western blotting. 50 ng of TliA was loaded in the middle of the gel as a reference. Two other Western blottings were obtained from different culture samples. All of the unpresented results exhibit similar patterns. Below the images, there are Western blottings of the same samples but with primary antibody against cytosolic Neo, the neomycin/kanamycin phosphotransferase 2 protein. The nonspecific lysis or leakage is minimal in all samples except capsid.

[0152] These non-secreted proteins have a relatively high theoretical pI. All of them (with one exception, CTP-TliA) were above 5.5, being either positively or less negatively charged. In contrast, the secreted proteins were relatively acidic and highly negatively charged with a pI that does not exceed 5.5 (FIG. 2a and FIG. 2b).

[0153] FIG. 2a and FIG. 2b shows correlation between % secretion of the target proteins and their pI values. The pI value of the target proteins is calculated from the sequence. The proteins that have not been secreted have their bars colored red. AP, EGF, and PLA1 are proven to be secreted in previous studies and are added in this figure. FIG. 2b added secretion percentage to pI. Three different biological replicates (independent culture samples) of the experiment in FIG. 1 were used for the quantitative analysis. Two highly basic outlier proteins that were not secreted, MAP (pI=9.61) and capsid (pI=9.25), were excluded from the plot. There was a negative correlation between the protein pI and their % secretion.

[0154] As could be seen in FIG. 1b and FIG. 2b, the secretion of NKC-TliA and CTP-TliA decreased dramatically from that of original TliA. These are derivatives of TliA with an N-terminally attached short, extremely positively-charged sequence (Table 2). CTP-TliA was not secreted at all. Note that CTP has nine consecutive residues composed solely of arginine with only one exception, alanine (RRARRRRRR), as described in Table 2.

[0155] Then, after quantification of western signal strengths of proteins, the percentage of secreted protein versus the total amount of expressed protein was plotted. The result was shown in FIG. 2b.

[0156] As shown in FIG. 2b, there seemed to be a weak negative correlation between protein pI and their secretion efficiency, but there were also a few exceptions.

[Example 7] Analysis of Lunasin and its Derivatives

[0157] Lunasin is an anticancer polypeptide from soybean Glycine max. It has a unique feature of nine consecutive aspartate (aspartic acid, Asp) sequences at its C terminus. The present inventors have constructed multiple derivatives of lunasin with different lengths of the aspartate polypeptide tails.

[0158] Then, lunasin and its derivatives were introduced to P. fluorescens via pDART, and their expression and secretion were observed via Western blotting, and the result was shown in FIG. 3a and FIG. 3b.

[0159] FIG. 3a and FIG. 3b is the result of confirming expression and secretion of lunasin and its derivatives with different lengths of the oligo-aspartic acid tails, to determine the optimal length of the oligo-aspartic acid sequence in P. fluorescens expression and secretion system.

[0160] FIG. 3a detected expression of lunasin and its derivatives in the cell and supernatant via Western blotting, and specifically, 36-l eq of cell extract and supernatant were loaded onto the gel and were analyzed via Western blotting. FIG. 3b represents protein sequence and domain structure of lunasin and its derivatives whose length of the aspartic acid tail is modified, and they were named as lunasin-D0, lunasin-D5, original lunasin (D9), lunasin-D15, and lunasin-D20, respectively.

[0161] As shown in FIG. 3a, the original lunasin showed that the highest secretion and relative amount of secreted proteins declined as the length of the oligo-aspartate tail decreased. The present inventors have also observed decreased secretion and expression levels in lunnasin-D15. Lunasin-D20 was not expressed in either the cell or supernatant. The exact sequence of the lunasin polypeptide and its derivatives is given in FIG. 3b.

[0162] Based on this experiment, the present inventors determined that the optimal length of the aspartate polypeptide sequence would be approximately nine, and we set up the experiments below.

[Example 8] Construction of pFD10 and pBD10 with Added Aspartate Polypeptide

[0163] Among the 20 most common amino acids, aspartic acid has the lowest side chain pKa value (Mathews, C. K. (2013) Biochemistry, 4th ed., Pearson, Toronto). Inspired by the lunasin protein sequence in the Example 7, the present inventors developed two plasmids that add the aspartate polypeptide sequence to the inserted proteins as well as the LARD3 signal sequence.

[0164] The present inventors have synthesized an aspartate-decamer-coding DNA sequence based on the DNA sequence of the lunasin gene's aspartate polypeptide tail, and have named D10 (DDDDDDDDDD: SEQ ID NO: 33). Then, the present inventors conjugated D10 to the pDART plasmid, creating two types of plasmid that either add D10 to the N terminus or to the C-terminus of the gene inserted to MCS, respectively, by inserting into pDART plasmid (named pGD10 and pBD10, respectively), and this was shown in FIG. 4.

[0165] FIG. 4 shows the structures of plasmids used, and represents the structure of pDART plasmid comprising MCS. tliD, tliE, tliF, and the LARD3-attached fusion protein are controlled in a single operon. (A) MCS is directly followed by the LARD3 gene, and thus the inserted target gene is expressed with LARD3 attached on its C terminus. (B) represents structure of pFD10 plasmid that attaches D10 sequence at the N terminus. The D10 gene directly follows the start codon and is located right before the MCS and LARD3. (C) represents structure of the pBD10 plasmid, which attaches D10 sequence at the C-terminal side, but before LARD3. The D10 gene is located between the MCS and LARD3.

[0166] Then, selected proteins were inserted into both of the newly created plasmids, pFD10 and pBD10. These pFD10 or pBD10-cloned recombinant proteins were introduced to P. fluorescens alongside their pDART-cloned counterparts, and the secretion efficiency was analyzed via Western blot analysis.

[Example 9] Insertion of TliA-Derived Recombinant Proteins into pFD10 and pBD10

[0167] NKC-TliA and CTP-TliA are both derivatives of TliA, each with an N-terminal basic-peptide attachment. Their secretion efficiency through TliDEF is significantly smaller than wild-type TliA (FIG. 1b and FIG. 10a, b).

[0168] FIG. 10a shows the result of enzyme plate assay of TliA, CTP-TliA and NKC-TliA, and TliA was secreted as expected (TliA is the natural substrate for the TliDEF transporter). However, secretion of CTP-TliA is blocked, and secretion of NKC-TliA is somewhat weaker than TliA.

[0169] FIG. 10b represents the result of western blotting of TliA, CTP-TliA, and NKC-TliA, and as could be seen in the enzyme plate assay, the secretion of TliA is strong, NKC-TliA is only weakly secreted, and CTP-TliA is not secreted. NKC is highly positively charged, and CTP has even more positively charged. CTP carries a consecutive nine residues composed solely of arginine with one exception in the middle, alanine.

[0170] However, the oligo-aspartate attachment on them by pFD10 or pBD10 greatly re-increases their secretion. The experimental result was shown in FIG. 5.

[0171] FIG. 5 shows the result of adding 10 aspartic acids to the N-terminus (1-D10) and C-terminus (BD10) of two kinds of TliA lipases in which NKC and CTP sequences are attached, respectively (NKC-TliA, CTP-TliA) and expressing through pDART plasmid, then detecting by western blotting and lipase activity measuring medium.

[0172] In (A) of FIG. 5, secretion strongly improved in both pFD10 and pBD10 when compared with pDART. (B) represents the result of enzyme plate assay of NKC-TliA in different plasmids. (C) is the result of western blotting of CTP-TliA in pFD10 and pBD10, and it is confirmed that secretion strongly increased in pBD10. (D) represents the result of enzyme plate assay of CTP-TliA in different plasmids. pBD10 exhibits a major increase in secretion. Two other Western blot results were obtained from different culture samples, and both of them exhibit similar patterns. Two other enzyme plate assays were obtained from different colonies, and both of them exhibit similar patterns.

[0173] In terms of the secretion ratio (secreted protein versus intracellular protein), NKC-TliA shows a dramatic increase in secretion after the addition of either an upstream or downstream D10 sequence, as shown in (A) and (B) of FIG. 5.

[0174] CTP-TliA also shows a drastic increase in secretion in both the Western blotting and activity plate assays when a downstream D10 sequence was added by pBD10, as shown in (C) and (D) of FIG. 5. In enzyme plate activity assays, the halo sizes of NKC-TliA and CTP-TliA in pDART or pBD10 are generally consistent with the band strength of the supernatant samples in their respective Western blotting results. However, pFD10 has a slightly smaller halo than expected from their band strength, indicating the possibility of a reduced enzymatic activity.

[Example 10] Insertion of Negatively-Charged Proteins to pFD10 and pBD10

[0175] A recombinant plasmid obtained by introducing genes for GFP, mannanase, maltose binding protein (MBP), and thioredoxin to pDART, pFD10, and pBD10 was introduced to P. fluorescens to prepare transformed proteins. The produced proteins were secreted by the TliDEF transporter, and the experimental result was shown in FIG. 6.

[0176] FIG. 6 shows secretion of negatively-charged proteins in pFD10 and pBD10. (A) represents the result of western blotting of GFP, and both pFD10 and pBD10 exhibit an increase in protein secretion in the supernatant. (B) represents the result of western blotting of Mannanase, and both pFD10 and pBD10 exhibit slight increases in mannase secretion. (C) represents the result of western blotting of MBP, and the increased secretion ratio was observed in both pFD10 and pBD10. (D) represents the result of western blotting of thioredoxin, and the signals were weak overall, but there was an increase in the secretion for both pFD10 and pBD10. Overall, the bands of more negatively-charged proteins in pBD10 appeared in slightly upward-shifted positions. Three other Western blot results for pDART and pBD10 were from different culture samples were obtained, whereas there were two other Western blot results for pDART and pFD10. All of them exhibit similar patterns.

[0177] As shown in FIG. 6, GFP showed the most dramatic increase in the increase of secretion. A comparison of the band strength of pDART and pBD10-inserted GFP showed a remarkable change in the supernatant versus cell expression ratio. pFD10-GFP also exhibited some improvement in terms of the ratio between the supernatant and the cell pellet ((A) of FIG. 6).

[0178] The case of mannanase was somewhat vague, but it could be concluded that pBD10-mannanase exhibits a better secretion than pDART-mannanase. In addition, although the absolute expression itself decreased, there was a small improvement in the ratio when an upstream D10 sequence was added by pFD10 ((B) of FIG. 6).

[0179] The secretion of MBP improved in both pFD10 and pBD10 in terms of the supernatant/cell ratio, compared with pDART ((C) of FIG. 6).

[0180] In the case of Trx (thioredoxin), the supernatant/cell ratio improved in pFD10 and pBD10 ((D) of FIG. 6).

[0181] Consequently, as the result of western blotting, it was confirmed that proteins in which aspartic acid was added to the N-terminus or C-terminus had increased (Supernatant) to intracellular (Cell) protein concentration ratio, and FD10 showed a significantly reduced pattern of expression.

[Example 11] Addition of Positively Charged Amino Acid OligomersConstruction and Analysis of pBR10

[0182] The present inventors constructed an additional plasmid that closely resembles pBD10, but with one difference. In this plasmid, the D10 sequence, the DNA sequence that codes for aspartate oligomer, was replaced with R10 that codes for arginine oligomer.

[0183] The present inventors inserted the TliA and GFP gene to pDART, pBD10, and pBR10 plasmids and examined their secretion by enzyme activity media (TliA only) and Western blotting, and the results were shown in FIG. 7.

[0184] FIG. 7 represents the result of adding 10 aspartic acids (D) or 10 arginines, respectively, at the C-terminus of TliA lipase and green fluorescent protein (GFP) and then detecting by western blotting and lipase activity media. negatively-charged proteins, TliA and GFP, were inserted in the plasmids that attach nothing except the signal sequence (pDART), oligo-aspartate (pBD10), and oligo-arginine (pBR10). A of FIG. 7 represents the result of western blot of TliA in these plasmids. TliA in pDART and pBD10 shows good secretion. However, the secretion was blocked when R10 was attached. B of FIG. 7 represents the result of enzyme plate assay of TliA in these plasmids. Secretion of TliA was blocked when it was inserted to pBR10.

[0185] In Western blotting of TliA, pDART and pBD10 exhibited good secretion efficiency. pBR10, however, blocked the secretion (A of FIG. 7). Similar patterns were observed in enzyme plate assay, pBR10 did not exhibit halo, but the others did (B of FIG. 7). In Western blotting of GFP, both pDART and pBD10 exhibited secretion. Yet again, pBR10 blocked secretion of GFP as it did to TliA (C of FIG. 7).

[Example 12] Western Blot Analysis of Supercharged Proteins

[0186] Green fluorescent protein (GFP) and its two supercharged derivatives, GFP (-30) and GFP (+36) by Average Number of Neighboring Atoms Per Sidechain Atom (AvNAPSA) (Lawrence M S, Phillips K J, Liu D R. Supercharging Proteins Can Impart Unusual Resilience. Journal of the American Chemical Society 2007; 129: 10110-10112.) method, were recombined with LARDS through pDART and introduced to P. fluorescens tliA prtA, to express proteins, and then the samples were analyzed via western blotting, and the result was shown in FIG. 8.

[0187] FIG. 8 represents secretion of GFP and supercharged GFPs. GFP (-30) exhibited a much higher extracellular (Supernatant) to intracellular (Cell) protein concentration ratio, and a significantly higher secretion than the original GFP.

[0188] On the other hand, positively supercharged GFP (+36) was detected in cells at a high concentration, but was not secreted at all outside of the cell (Supernatant). Although the bands of the supercharged GFPs are also slightly shifted upwards, but two other Western blot results from different culture samples were obtained, and both of them exhibit similar patterns.

[0189] As could be seen in FIG. 8, both GFP and GFP (-30) were detected in the cell pellet and the supernatant, indicating that they were effectively expressed and secreted to the extracellular space. Herein, it could be confirmed that GFP (-30) was more strongly localized to the supernatant than the original GFP.

[0190] In contrast, GFP (+36) was heavily expressed but localized in the cell pellet and was not secreted to the extracellular space. The pI values for these recombinant proteins were 4.64 for GFP (-30), 5.36 for unmodified GFP, and 10.42 for GFP (+36).

[Example 13] Confirmation of the Optimal Linker Length for Increasing the Protein Secretion Efficiency

[0191] NKC-TliA was selected as a model protein. NKC consists of 21 amino acids, and is a peptide forming amphiphilic -helix. 21 amino acids include lysine a lot, and as pI=10.78, when NKC-TliA is prepared by fusion to TliA lipase in which pI=5.01, pI=5.34 and the protein secretion is reduced.

[0192] The present inventors have confirmed that the secretion by replacing all the lysine of NKC protein to aspartate (NKC(-)), and in addition, have compared the secretion efficiency of NKC(-) through various linker lengths by linking linkers with various lengths to NKC(-) and TliA through western blotting and activity analysis plate, and the results were shown in FIG. 15. The lengths of linker were represented by L1 with one GGGGS from NKC (-) where noting is present, L2 with 2, and L3 with 3.

[0193] FIG. 15 represents the protein secretion in TliA, NKC(-), NKC-L1, NKC-L2, NKC-L3, and NKC-TliA. (A) of FIG. 15 is the western blotting result of TliA, and (B) of FIG. 15 shows the result of enzyme plate analysis of TliA in other plasmids.

[0194] As the result of the western blotting of (A) of FIG. 15, it could be confirmed that the far right NKC-TliA was not secreted at all, but secretion of NKC(-) in which all the lysine was replaced with aspartate and the protein in which a linker was attached to negatively charged NKC was significantly increased.

[0195] According to the result of the activity analysis plate of (B) of FIG. 15, it could be confirmed that the protein secretion was increased in NKC(-) than the conventional NKC, and the secretion was increased overall when a linker was introduced, and in particular, when 3 linkers were attached, the secretion was significantly increased. Through this result, it could be seen that the negatively charged NKC increased the protein secretion.

[Example 14] Increased Protein Secretion by Negative Supercharge

[0196] The present inventors have observed the tendency of protein secretion by replacing amino acids of proteins to negatively charged amino acids, to confirm secretion efficiency changes by changing the protein charge.

[0197] For this, negative charge supercharge 10 and positive charge supercharge+13 were produced from Streptavidin (SAV) wild type protein, and similar to this, supercharge proteins with the negative charger supercharge 20 and positive charge supercharge+19 were produced from glutathione S-transferase (GST), to analyze the protein secretion, and the result was shown in the following Table 16.

[0198] FIG. 16 represents the analysis result of secretion of 10SAV, wtSAV, +13SAV and 20GST, wtGST, and +19GST (SAV: streptavidin/GST: glutathione S-transferase). SAV (135aa) produces a tetramer and GST (215aa) produces a dimer. In gene synthesis, the charge of monomers was calculated (-10SAV: pI4.96/wtSAV:pI6.76/+13SAV: pI10.29/-20GST: pI4.73/wtGST: pI7.86/+19GST: pI9.87).

[0199] As shown in FIG. 16, it could be confirmed that negatively charged supercharger proteins were present in cells a lot and they were secreted well, but it could be seen that the wild type protein and positively charged supercharge proteins were not expressed and secreted, and it could be confirmed that the negative charge supercharge increased the protein secretion.

[0200] Similarly, while looking at the structure randomly without using AvNAPSA method, supercharged glutathione S-transferase (GST) and streptavidin (Say), in which protruding amino acids were replaced with aspartic acid or arginine, were added to pDART and were expressed, to perform western blotting, and the result was shown in FIG. 17.

[0201] As FIG. 17, it could be seen that the extracellular (Supernatant) to intracellular (Cell) protein concentration ratio of negatively supercharged proteins (indicated by red) was remarkably increased. On the other hand, positively supercharged proteins were detected in cells at a significantly high concentration, but they were not detected or were detected at a low concentration outside of cells (Supernatant).

[Example 15] Confirmation of Extracellular Secretion Increase of Negatively Supercharged Protein Using AvNAPSA Method

[0202] MelC.sub.2 tyrosinase, cutinase (Cuti), Chitinase (Chi), and M37 lipase were negatively sugercharged by AvNAPSA method, and then negatively supercharged proteins (red) and non-supercharged natural proteins corresponding thereto (black) were added to pDART plasmids, respectively, and were expressed to detect them by western blotting, and the result was shown in FIG. 18.

[0203] Specifically, the negatively supercharging method using AvNAPSA is as follows. At first, arpartic acid and glutamic acid are replaced and enter to a suitable position to obtain the negatively supercharged protein sequence by AvNAPSA algorithm (1. Lawrence M S, Phillips K J, Liu D R. Supercharging Proteins Can Impart Unusual Resilience. Journal of the American Chemical Society 2007; 129: 10110-10112.). Then, the DNA sequence corresponding to the protein sequence was synthesized, and the synthesized DNA sequence was added to pDART plasmid and then negatively supercharged proteins were prepared.

[0204] It could be seen the negatively supercharged proteins were observed not only inside of cells (C) but also outside of cells (S) at a very high concentration, different from natural proteins which were not detected at all outside of cells, and their secretion were remarkably increased. In case of MelC2 tyrosinase protein, small sequence differences including His-tag result in small size differences between supercharged proteins and natural proteins.

[0205] In other words, through the experiment, the present inventors have confirmed that proteins which were not secreted in the past could be extracellularly secreted with considerable efficiency, by supercharging proteins such as tyrosinase, cutinase, and the like, to which the secretion production method was not applicable by conventional techniques, using AvNAPSA algorithm.

[Example 16] Confirmation of TliA Protein Secretion in Cells in which T1SS Transporters Isolated from Three Different Kinds of Bacteria are Expressed

[0206] 16-1. Escherichia coli HlyBD+TolC, Dickeya dadantii PrtDEF, Pseudomonas aeruginosa AprDEF Isolation

[0207] The present inventors amplified certain part of operon comprising HlyB, HlyD genes from isolated genome of Escherichia coli CFT073 strain (Genbank AE014075) through PCR using two primers of hlyBD-s (SEQ ID NO: 34: GGGGAGCTCGGATTCTTGTCATAAAATTGATT), hlyBD-a (SEQ ID NO: 35: GGGGGATCCTTAACGCTCATGTAAACTTTCT), and plasmid pSTV-HlyBD was prepared in which this was inserted in order together with start codon and kozak sequence to pSTV plasmid (one of derivatives of pACYC plasmid) by amplifying transporter genes from genome of each strain through PCR, respectively. TolC consisting of transporters together with HlyB and HlyC was not comprised separately, since it is produced by E. coli itself.

[0208] In addition, the present inventors prepared the plasmid expressing three genes of PrtD, PrtE and PrtF of Dickeya dadantii, pEcPrtDEF (Delepelaire P, Wandersman C Protein secretion in gram-negative bacteria. The extracellular metalloprotease B from Erwinia chrysanthemi contains a C-terminal secretion signal analogous to that of Escherichia coli alpha-hemolysin. J Biol Chem. 1990; 265:17118-17125) and the plasmid expressing three genes of AprD, AprE and AprF of Pseudomonas aeruginosa, pAGS8 (Duong F, Soscia C, Lazdunski A, Murgier M. The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system. Mol Microbiol. 1994; 11:1117-1126).

[0209] 16-2. Confirmation of Protein Secretion in Cells in which T1SS Transporters Isolated from Three Different Kinds of Bacteria are Expressed

[0210] The present inventors introduced one plasmid which the gene of TliA protein (original substrate of TliDEF transporter) was inserted to pQE184 plasmid, and one of plasmids expressing one kind of T1SS transporters isolated from three different kinds of bacteria prepared above (namely, pSTV-HlyBD expressing Escherichia coli HlyBD, pEcPrtDEF expressing Dickeya dadantii PrtDEF, pAGS8 expressing Pseudomonas aeruginosa AprDEF) to E. coli by heat shock method simultaneously and expressed TliA and one of three transporters simultaneously, and then measured secretion of the recombinant target proteins from lipase enzyme activity measuring media to outside of cells through color changes of colony peripheral media, and the result was shown in FIG. 19.

[0211] As shown in FIG. 19, it could be confirmed that all the three transporters of Escherichia coli HlyBD+TolC (E. coli expresses the original TolC protein), Dickeya dadantii PrtDEF, and Pseudomonas aeruginosa AprDEF secreted TliA protein successfully. This can be inferred from the fact that halo is not observed in the strain in which only TliA protein is expressed (TliA only) without further expression of transporter proteins in E. coli. The result means that T1SS proteins of Escherichia coli, Dickeya dadantii, and Pseudomonas aeruginosa other than Pseudomonas fluorescens can recognize the LARD3 signal sequence of TliA.

[Example 17] Confirmation of Cutinase Protein Secretion in Cells in which T1SS Transporters Isolated from Three Different Kinds of Bacteria are Expressed

[0212] 17-1. Preparation of Negatively Supercharged Cutinase Protein

[0213] Negatively supercharged cutinase protein (Cuti(-)) was prepared using AvNAPSA method to cutinase protein (Cuti).

[0214] 17-2. Confirmation of Cutinase Protein Secretion in Cells in which T1SS Transporters Isolated from Three Different Kinds of Bacteria are Expressed

[0215] After attaching the LARD3 signal sequence by the method of inserting cutinase genes to pLARD3 plasmid in which the gene of LARD3 signal sequence was inserted right behind of the multiple cloning site, based on pUC19 plasmid, to cutinase protein and negatively supercharged cutinase protein, with the plasmid expressing three different kinds of T1SS transporter proteins (Escherichia coli HlyBD+TolC, Dickeya dadantii PrtDEF, Pseudomonas aeruginosa AprDEF) obtained by the method of Example 16, similar to the method of Example 16, two plasmids were introduced to E. coli cells simultaneously and were expressed simultaneously, and they were culture in cutinase enzyme activity measuring media at 37 C. for 3 days, and then the protein secretion to outside of E. coli was measured through color changes of colony peripheral media, and the result was shown in FIG. 20.

[0216] As shown in FIG. 20, it could be observed that the secretion of negatively supercharged cutinase was remarkably high than non-negatively-supercharged cutinase, in all the three kinds of T1SS transporter proteins. In the same manner, it could be inferred through comparison to control groups in which an empty plasmid was added instead of the transporter plasmid (Cuti(-) only, Cuti only).

[Example 18] Confirmation of Extracellular Secretion of Cutinase Protein Using Western Blotting

[0217] After attaching the LARD3 signal sequence to cutinase protein (Cuti) and negatively supercharged cutinase protein (Cuti(-)), they were expressed in E. coli with three different kinds of T1SS transporter proteins obtained by the method of Example 16 and were liquid cultured, and then the intracellular and extracellular protein concentration was detected by western blotting, and the result was shown in FIG. 21.

[0218] As shown in FIG. 21, it could be observed that the secretion of negatively supercharged cutinase was remarkably high than non-negatively-supercharged cutinase, in all the three kinds of T1SS transporter proteins. In the same manner, the secretion fact could be inferred by comparison to control groups in which an empty plasmid was added instead of the transporter plasmid (Cuti(-) only, Cuti only).

[Example 19] Confirmation of Extracellular Secretion of M37 Lipase Protein Using Western Blotting

[0219] After attaching the LARD3 signal sequence to M37 lipase protein and negatively supercharged M37 lipase protein (M37(-)), they were expressed in E. coli with three different kinds of T1SS transporter proteins obtained by the method of Example 16 and were liquid cultured, and then the intracellular and extracellular protein concentration was detected by western blotting, and the result was shown in FIG. 22.

[0220] As shown in FIG. 22, it could be observed that the secretion of negatively supercharged M37 was remarkably high than non-negatively-supercharged M37, in all the three kinds of T1SS transporter proteins. In the same manner, the secretion fact could be inferred by comparison to control groups in which an empty plasmid was added instead of the transporter plasmid (M37(-) only, M37 only).

[Example 20] Evaluation of Sequence Identity of T1SS ABC Transporters

[0221] The sequence identity of TliD of TliDET transporter of Pseudomonas fluorescens and T1SS ABC transporters of Escherichia coli HlyBD+TolC, Dickeya dadantii PrtDEF, and Pseudomonas aeruginosa AprDEF with ABC proteins of other kinds of T1SS transporters was measured, and the result was shown in FIG. 23. FIG. 23 represents the sequence identity between TliDEF transporter and various T1SS transporters, and the proportion occupied by the sequence-like portion in the entire sequence.

[0222] Specifically, the sequence identity between transporter proteins was calculated using NCBI BLASTp algorithm, and the indicated sequence identity was calculated by omitting some sequences that greatly differ from each other according to the normal calculation method of the algorithm, and limiting within the query coverage. As a result, the omitted sequence portion was less than 10% in any case, suggesting that the sequence identity was very reliable.

[0223] The sequence identity of TilD of TliDEF transporter with various T1SS ABC transporters varied from relatively high to relatively low. Among them, the sequence identity of three T1SS transporters of AprD, PrtD, and HlyB which were examples in Examples 16, 17, 18 and 19 were exhibited as 60%, 59%, and 27%, respectively.

[0224] Accordingly, the present inventors confirmed that the protein secretion enhancement technology of negatively supercharging was not limited to Pseudomonas fluorescens microorganism TliDEF transporters, and could be widely applied to various T1SS transporters having about 27% of the amino acid sequence identity (homology).

[Example 21] the Preparation of the Target Protein with Lowered pI by Substituting with Neutral Amino Acids

[0225] As described above, negatively supercharging the target protein to enhance the secretion came along with the problem of losing the enzymatic activity in some cases. To deal with this problem, The inventors devised a method to make the substitution less damaging to the protein's overall fold, which was replacing these solvent-exposed positively charged residues to the neutral hydrophilic amino acids, not negatively charged ones. The inventors primarily used relatively bulky glutamine to replace arginine and lysine, as both of them were quite bulky amino acids. This way, the genes encoding the two proteins MelC2(Q) and M37(Q) was prepared. The (Q) parts designate that the solvent-exposed amino acids were replaced with glutamine (single-letter code Q).

[0226] Further on, the inventors refer to the technique as Superneutralization or Superneutralizing which is a process of removing charges by replacing charged residues on the solvent-exposed surface of a protein or protein complexes with neutrally-charged amino acids. The term selective superneutralization of positive charges is used to describe a process of superneutralization applied specifically and solely on positively charged residues so that the mutated protein mainly consists of negative charge. It turned out that removing positive charges via superneutralization (not adding any additional negative charge) also improves the secretion of proteins dramatically, in both MelC2 and M37 (Error! Reference source not found.A). Besides, the superneutralized M37(Q) exhibited enzymatic activity, as can be seen from the plate activity assay (Error! Reference source not found.C).

[0227] More specifically, in FIG. 23A, the solvent-accessible positively charged residues were replaced with glutamine (single-letter code Q), a neutral hydrophilic amino acid. The two resultant proteins, MelC2(Q) and M37(Q) were highly localized to the culture supernatant, compared to their wild-types. The negatively supercharged versions of these two proteins, MelC2(-40) and M37(-23) were also loaded as a comparison.

[0228] In FIG. 23C, the colonies of P. fluorescens cells expressing the M37 derivatives were streaked on the LB agar plate supplemented with 0.5% colloidal glyceryl tributyrate. The lipase activity of the secreted M37 lipase creates a visible clear halo around the streaks. As a negative control, P. fluorescens cells harboring pDART-GFP plasmid was streaked as well. Negatively supercharged M37 variants, M37(-23) and M37(-14) had significantly lower halo size compared to the wild-type M37. Especially, M37(-23) exhibited little enzymatic activity even though it was mainly localized in the extracellular space. On the other hand, however, M37(Q) and M37(var) had large halo size, comparable or even larger than the wild-type.

[0229] The inventors did not test the activity of MelC2(Q), mainly because the MelC2 protein itself is generally not active without its caddie protein MelC1, which is not present in the P. fluorescens expression host when introduced via pDART plasmid. It might be possible to examine the activity of pDART-inserted MelC2(Q) by co-expressing MelC1 in P. fluorescens, but that experiment was not performed here. Comprehending the results, the superneutralization approach was proven to be a better option than the conventional supercharging method in terms of the preservation of protein activity, while being just as effective in terms of the secretion enhancement.

[0230] Specifically, the computational designing of supercharged or superneuturalized proteins were performed. The inventors utilized the AvNAPSA algorithm to boost the productivity and reproducibility of supercharged protein designing. The AvNAPSA method, an abbreviation for Average Neighbor Atoms per Sidechain Atom was developed by Liu group to automatically design supercharged proteins, in their pursuit of making a resilient folded protein and for animal cellular protein targeting. In this paper, however, the supercharging protocol is used to generate proteins that are compatible with ABC transporter secretion. AvNAPSA algorithm automatically scores the residues according to the exposure to the external space, rather than the facing other parts of the protein. More specifically, it calculates the number of atoms within a certain distance from each atom of the side chain and returns the AvNAPSA score for each residue. The lower the score, the more exposed the residue to the solvent. We gradually mutated positively charged residues in the increasing order of AvNAPSA score until the level of the mutation was similar to the level of mutation we would have if we performed manual supercharging. The exact AvNAPSA thresholds for our mutated proteins are given in Table 5. Also note that the inventors excluded any residue proximal (closer than seven residues apart) to the active site residues.

TABLE-US-00005 TABLE 5 Full name Abbrev. (SEQ ID NO) Source Source type M37 M37 lipase Photobacterium lipolyticum Genomic DNA (SEQ ID NO: 11) M37(23) M37, 23 negatively AvNAPSA supercharging, Synthesized supercharged threshold = 100 (SEQ ID NO: 36) M37(14) M37 lipase, 14 negatively AvNAPSA supercharging, Synthesized supercharged threshold = 90 (SEQ ID NO: 37) M37(Q) M37, selectively Manually superneutralized Synthesized superneutralized (SEQ ID NO: 38) M37(var) M37, randomly mutated Random mutation and activity Synthesized via and screened screening mixed-base (SEQ ID NO: 39)

[0231] Synthesizing and Cloning the Gene of Variationally Supercharged M37

[0232] The inventor prepared the DNA sequence of variationally supercharged M37 lipase, which we named M37(var), by replacing the codons for the surface-exposed positively charged amino acid residues with the degenerate codon. For example, the IUPAC DNA code R denotes the purine base, which is guanine or adenine. The inventor aimed to find the Goldilocks variant somewhere between M37(-14), which had enzymatic activity but was not secreted, and M37(-23), which was secreted but had no enzymatic activity. The inventor examined the amino acid sequences of M37(-23) and M37(-14) and marked the residues where the two differed. Then, the inventors replaced codons for those residues with the degenerate codons. For instance, the residue Lys36 of M37 lipase was replaced by glutamic acid in M37(-23) but remained unchanged in M37(-14). Therefore, the inventor placed the degenerate codon RAG at that position. For the degenerate codon RAG, there were two possible outcomes: GAG, which codes for glutamic acid; and AAG, which codes for lysine. The inventors would have preferred to use the set glutamic acid or arginine, but such a combination was impossible. Therefore, the inventors used the RAG codon in place of these residues as well. The exact sequence we ordered for the construction of M37(var) is given in Supplementary Section A. After the DNA sequence was designed, the inventor used the DNAWorks web server (https://hpcwebapps.cit.nih.gov/dnaworks/) to convert the sequence into a set of synthesizable primers. The used parameters were as the following: oligo length 58 nucleotides, annealing temperature 62 C., oligo concentration 1.0010.sup.7 M, Na.sup.+/K.sup.+ concentration 0.05 M, Mg.sup.2+ concentration 0.002 M, number of solutions 1, no TBIO mode (PTDS mode was used instead). Then, the inventors manually examined the output oligos and made sure that no degenerate codon was present at the end of any overlapping region between oligos. The inventors ordered the oligos from Cosmogenetech, and assembled them using the PCR-based DNA synthesis method described in a previous publication. The obtained PCR product was purified, restricted, and then introduced into pDART plasmid like the rest of the genes handled in the example.

[Example 22] the Preparation of the Target Protein with Lowered pI by Mutation and Activity Screening Method

[0233] Each residue modification induces some change in the protein's 3-dimensional structure, but the amount of the change varies. Some residue mutations may barely affect the overall structure except for its side chain itself, while others could accompany a serious distortion in the secondary and/or tertiary structure. However, we cannot yet accurately evaluate the magnitude of structural change induced by each point mutation, since we are mutating multiple residues at once. So, our idea was to leave the evaluation to the cells, not the human researchers. We synthesized the gene of M37 lipase, with the random mutations. Specifically, the solvent-exposed positively charged amino acids (arginine and lysine) were randomly mutated into negatively charged or neutral hydrophilic amino acids. We used mixed-base DNA synthesis to randomly choose codons between the two amino acids. After the randomly mutated genes were synthesized, they were incorporated into the pDART plasmid, and then introduced into E. coli. We screened for the clone that exhibited the most prominent halo in the lipase activity assay LB agar plate. The resulting clone was then characterized via DNA sequencing. The resulting clone had both excellent secretion (Error! Reference source not found.B) and activity (Error! Reference source not found.C). The mixed-base strategy utilized to prepare M37(var) is given in Error! Reference source not found.D.

[0234] More specifically, in FIG. 23B, the solvent-accessible positively charged or neutral hydrophilic residues of M37 lipase were randomly mutated into neutral or negatively charged amino acids. Then, the clone which exhibited the largest halo on the activity plate assay was selected and sequenced. The inventors named this mutant lipase M37(var). The inventors analyzed the secretion of M37(var) via western blot. It turned out that it was indeed secreted well. FIG. 23D shows the mixed-based codon strategy utilized to prepare M37(var) mutant.