SITE-SPECIFIC MUTAGENESIS MODIFIED YEAST DIPEPTIDYL PEPTIDASE III
20190246664 ยท 2019-08-15
Assignee
Inventors
- Daling LIU (Guangzhou, Guangdong, CN)
- Dongsheng YAO (Guangzhou, Guangdong, CN)
- Xiyang WU (Guangzhou, Guangdong, CN)
- Chunfang XIE (Guangzhou, Guangdong, CN)
Cpc classification
A23L5/20
HUMAN NECESSITIES
International classification
Abstract
The invention relates to a site-specific mutagenesis modified yeast dipeptidyl peptidase III. The yeast dipeptidyl peptidase III is an isolated mutant produced by making a plurality of amino acid substitutions in a wild type yeast dipeptidyl peptidase III derived from Saccharomyces cerevisiae S288c having an amino acid sequence of SEQ ID NO. 1, said amino acid substitutions comprising substitutions at positions 570, 572 and 574, so that the site-specific mutagenesis modified yeast dipeptidyl peptidase III is an enzyme having an oxidative decomposition activity on 6-methoxy-bifuran coumarin. The site-specific mutagenesis modified yeast dipeptidyl peptidase III can be used in preparation of a feed and additives thereof, and a food and additives thereof, in which 6-methoxy difuran coumarin is eliminated, and can be used in preparation of a medicament for preventing diseases induced by 6-methoxy-difuran coumarin.
Claims
1. A site-specific mutagenesis modified yeast dipeptidyl peptidase III, wherein the yeast dipeptidyl peptidase III is an isolated mutant produced by making a plurality of amino acid substitutions in a wild type yeast dipeptidyl peptidase III derived from Saccharomyces cerevisiae S288c having an amino acid sequence of SEQ ID NO. 1, said amino acid substitutions comprising substitutions at positions 570, 572 and 574, so that the site-specific mutagenesis modified yeast dipeptidyl peptidase III is an enzyme having an oxidative decomposition activity on 6-methoxy-bifuran coumarin.
2. The site-specific mutagenesis modified yeast dipeptidyl peptidase III according to claim 1, wherein the amino acid substitution at position 570 is a Alanine (Ala, A) amino acid residue for a Lysine (Lys, K) amino acid residue, the amino acid substitution at position 572 is a Lysine (Lys, K) amino acid residue for a Glycine (Gly, G) amino acid residue, and the amino acid substitution at position 574 is a Histidine (His, H) amino acid residue for a Tryptophan (Trp, W) amino acid residue, said site-specific mutagenesis modified yeast dipeptidyl peptidase III having an amino acid sequence of SEQ ID NO. 2.
3. An isolated DNA molecule encoding a site-specific mutagenesis modified yeast dipeptidyl peptidase III according to claim 2.
4. The isolated DNA molecule according to claim 3, wherein the DNA molecule comprises a nucleotide sequence of SEQ ID NO. 3.
5. A recombinant expression vector comprising a DNA molecule according to claim 3.
6. A host cell comprising a DNA molecule according to claim 3.
7. A method for producing a site-specific mutagenesis modified yeast dipeptidyl peptidase III according to claim 1, said method comprising: cultivating the transformed host cell according to claim 6 under conditions suitable for expression of the dipeptidyl peptidase III; and separating, purifying and recovering the mutant yeast dipeptidyl peptidase III.
8. A use of the site-specific mutagenesis modified yeast dipeptidyl peptidase III according to claim 1 in preparation of a feed and additives thereof, and a food and additives thereof, in which 6-methoxy difuran coumarin is eliminated.
9. A use of the site-specific mutagenesis modified yeast dipeptidyl peptidase III according to claim 1 in preparation of a medicament for preventing diseases induced by 6-methoxy-difuran coumarin.
10. A recombinant expression vector comprising a DNA molecule according to claim 4.
11. A host cell comprising a DNA molecule according to claim 4.
12. A host cell comprising a recombinant expression vector according to the claim 11.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0018]
[0019]
DETAIL DESCRIPTION OF THE INVENTION
[0020] The terms used in the present disclosure, unless otherwise indicated, are the meanings commonly understood by those skilled in the art. Definitions of some special terms used in the invention are provided in the following.
[0021] The wtyDPP refers to a wild type yeast dipeptidyl peptidase III, and its gene is represented by an italicized wtyDPP.
[0022] The myDPP refers to a mutant yeast dipeptidyl peptidase III, and its gene is represented by an italicized myDPP.
Example 1: Synthetise of wtyDPP and myDPP
[0023] According to the invention, based on the gene sequence (NCBI Database, NM_001183312) of dipeptidyl peptidase from Saccharomyces cerevisiae S288C, 5-GTCGAATTC-3 is added at the 5 end, and 3-CCTAGGGAC-5 is added at the 3 end, GAATTC is a restriction site of EcoRI, and GGATCC is a restriction site of BamHI.
[0024] The gene wtyDPP is synthesized by an artificial all-synthesis method.
[0025] According to the invention, based on the gene sequence (NCBI Database, NM_001183312) of dipeptidyl peptidase from Saccharomyces cerevisiae S288C, the amino acid residue at position 570 is replaced by Alanine (Ala, A) amino acid residue, and the amino acid residue at position 572 is replaced by Lysine (Lys, K) amino acid residue, and the amino acid residue at position 574 is replaced by Histidine (His, H) amino acid residue. 5-GTCGAATTC-3 is added at the 5 end, and 3-CCTAGGGAC-5 is added at the 3 end, GAATTC is a restriction site of EcoRI, and GGATCC is a restriction site of BamHI. The gene myDPP is synthesized by an artificial all-synthesis method.
[0026] After the site-specific mutagenesis modification, Alanine (Ala, A) at position 570, Lysine (Lys, K) at position 572, Histidine (His, H) at position 574, and Glutamine (Gln, Q) at position 576, Histidine (His, H) at position 578, Methionine (Met, M) at position 579, Glutamine (Gln, Q) at position 580, Alanine (Ala, A) at position 581 and Arginine (Arg, R) at position 582 forms a sequence AXKXHXQXHMQAR, wherein A is Alanine, R is Arginine, X is any amino acid.
[0027] The gene synthesis is completed by a commercial company, for example, Shanghai Jierui Biological Co., Ltd.
Example 2: Construction of Recombinant Expression Vector for wtyDPP and myDPP
[0028] Gene cloning was carried out according to a conventional method (Sambrook, et al. 2001, molecular cloning a laboratory manual. Cold spring harbor laboratory press. USA). Genes wtyDPP and myDPP obtained from Example 1 were respectively cloned to an expression vector pHIL-S1 to build two recombinant expression vectors pHIL-S1-wtyDPP and pHIL-S1-myDPP. The cloned target genes were identified by restriction enzymatic incisions and sequencing.
[0029] Detailed Steps of the Method
[0030] The construction procedure of the recombinant vector pHIL-S1 containing myDPP was as following: EcoRI+BamHI double enzymatic incisions were made on the vector pHIL-S1 and the target segment myDPP, and the enzymatic incision product was isolated by 0.8% agarose gel electrophoresis, and then the product was cut from the gel and recovered. Let the vector pHIL-S1 connect with the myDPP by T4 DNA ligase. E. coli DH5a competent cells were prepared using CaCl.sub.2 method, and then were transformed by the recombinant vectors. The transformants were screened, and the recombinant vectors were extracted from the screened transformants. The recombinant vector pHIL-S1-myDPP was identified by EcoRI+BamHI, HindIII, SacI restriction enzymatic incisions. The vector DNA was extracted and purified form the recombinant vector by PEG purification method (Sambrook, et al 2001, Molecular Cloning A Laboratory Manual. Cold Spring Harbor Labroratory Press. USA). T7 and SP6 were used as sequencing primers, and the DNA was sequenced in forward and reverse directions by using DNA automatic sequencer. The result of the restriction enzyme digestion of the recombinant vector pHIL-S1-myDPP was shown in
[0031] The construction procedure of the recombinant vector pHIL-S1 containing wtyDPP was as following: the target segment myDPP was replaced by wtyDPP during the construction procedure of the recombinant vector pHIL-S1 containing myDPP, and the other operations is the same as that in the construction procedure of the recombinant vector pHIL-S1 containing myDPP.
Example 3: Expression of Recombinant myDPP and Recombinant wtyDPP
[0032] The expression of the recombinant myDPP was as follows: SacI restriction enzymatic incision was made on the recombinant vector pHIL-S1-myDPP and the vector pHIL-S1, the enzymatic incision product was isolated by 0.8% agarose gel electrophoresis, and then the product was cut from the gel. The linear recombinant vector pHIL-S1-myDPP and the vector pHIL-S1 were recovered. The pichia pastoris GS115 was transformed by a spheroplast method according to the handbook of Pichia Expression Kit (Invitrogen Inc., USA), and the Mut.sup.+ transformants were screened. The recombinant bacteria were induced to expression by using methanol as a unique carbon source, according to the operations of the handbook of Pichia Expression Kit. The result of SDS-PAGE electrophoresis shows that there was obvious target protein band appeared in the supernatant of the culture after the induced expression. And there was no target protein band appeared in the supernatant of the culture of the negative control bacteria containing empty vector after 96 hours under the same condition. The results were shown in
[0033] Detailed Steps of the Method
[0034] The homologous recombination of the recombinant and pichia pastoris
[0035] I. Linearization of the Vector
[0036] The recombinant vector pHIL-S1-myDPP and the vector pHIL-S1 were cleaved with SacI restriction enzyme. The linearized vector pHIL-S1 will be used as a control of the following experiments.
[0037] The enzymatic incision of pHIL-S1-myDPP (120 l total system): 12 l of Buffer L+8 l SacI+100 l/pHIL-S1-myDPP.
[0038] The enzymatic incision of pHIL-S1 (120 l total system): 12 l of Buffer L+8 l SacI+100 l pHIL-S1.
[0039] Samples were recovered by a 0.8% agarose gel electrophoresis, and then cut from the gel, and the recombinant vector pHIL-S1-myDPP and the vector pHIL-S1 were recovered.
[0040] II. Culture of Pichia Pastoris GS115 Used for Spheroplast Culture
[0041] The method comprises the following steps:
[0042] 1. a GS115 monoclone was selected from a flat plate and was inoculated into 10 ml YPD (Yeast Extract Peptone Dextrose medium). Culturing overnight at 30 C. in a shaking 150 ml conical incubator (250-300 rpm).
[0043] 2. respectively taking 5, 10, 20 l bacteria liquid from 10 ml overnight YPD culture and then inoculating into 200 ml YPD. Culturing overnight in a shaking 500 ml conical incubator (250-300 rpm).
[0044] 3. The three cultures were detected for OD600. The ones with OD600=0.2-0.3 were selected, and pelleted by centrifugation at 1500g for 5 minutes at room temperature. The supernatant was discarded. The collected cells were used for spheroplast transformation.
[0045] III. Preparation of Pichia pastoris GS115 Spheroplasts
[0046] 1. The cell pellet was re-suspended in 200 ml sterile water, and then transferred to two 10 ml sterile centrifuge tubes.
[0047] 2. The cells were pelleted by centrifugation at 1500g for 5 min at room temperature. The supernatant was discarded.
[0048] 3. The cell pellet was washed with fresh prepared SED, followed by centrifugation at 1500g for 5 min at room temperature. The supernatant was discarded.
[0049] 4. The cell pellet was washed with 1M Sorbitol solution, followed by centrifugation at 1500g for 5 min at room temperature. The supernatant was discarded.
[0050] 5. The cell pellet was re-suspended in 10 ml SCE.
[0051] 6. Zymolyase in a tube was thawed and mixed by flicking the tube.
[0052] 7. 7.5 l of Zymolyase was added into the cells and the cells were incubated for 30 min at 30 C.
[0053] 8. The cells were pelleted by centrifugation at 750g for 5 min at room temperature. The supernatant was discarded.
[0054] 9. The transformation mixture was washed with 1M Sorbitol solution, mixed by flicking the tube to disperse the precipitate. The cells were pelleted by centrifugation at 750g for 10 min at room temperature. The supernatant was discarded, and the cell pellet was collected.
[0055] 10. The cell pellet was washed with 10 ml CaS solution, followed by centrifugation at 750g for 5 min. The supernatant was discarded.
[0056] 11. The cell pellet was re-suspended in 0.6 ml CaS solution. The spheroplasts must be used within 30 min.
[0057] IV. Spheroplast Transformation of Pichia pastoris GS115
[0058] 1. Aliquots of 100 l each of Pichia pastoris GS115 spheroplasts were respectively transferred to three 15 ml sterile centrifuge tubes A, B and C.
[0059] 2. Tube A (no DNA) as negative control, tube B (added 30 l linearized vector pHIL-S1), tube C (added 30 l linearized recombinant plasmid pSA, incubated for 10 min at room temperature). 3 ml of fresh PEG/CaT was prepared at the same time.
[0060] 3. Aliquots of 1 ml each of fresh PEG/CaT were added to tubes A, B and C, mixed gently and incubated for 10 min at room temperature.
[0061] 4. The cells were pelleted by centrifugation at 750g for 5 min at room temperature. The supernatant was discarded.
[0062] 5. The cell pellets were re-suspended in 150 l SOS, incubated for 20 min at room temperature.
[0063] 6. Aliquots of 850 l 1 M Sorbitol solution each were added to the tubes. 7. The entire transformations were plated on RD solid incubation plates using a sterile spreader (200 l/plate). The plates were incubated at 28-30 C. Transformants were appeared between 4-6 days.
[0064] V. Selection of Mut.sup.+ Transformants
[0065] 1. Using a sterile toothpick, His+ transformants were patched on both MM and MD plates, the strains GS115/His.sup.+Mut.sup.s Albumin and GS115/His.sup.+Mut.sup.+-gal were also patched on the plates as controls.
[0066] 2. Plates were incubated at 28-30 C. for 2 days.
[0067] 3. After two days, scored both MM and MD plates. Mut.sup.+ strains will grow normally on both MM and MD plates, while Mut.sup.s will grow normally only on the MD plate but little or no growth on MM plate.
[0068] VI. Induced Expression of the Recombinant Strains
[0069] 1. Inoculated a single colony of His.sup.+Mut.sup.+ transformant in 25 ml BMG in a 250 ml baffled flask. Grew at 28-30 C. in a shaking incubator (250-300 rpm) until the culture reached OD600=2-6 (16-18 h).
[0070] 2. Cells were harvested by centrifugation at 1500-3000g for 5 min at room temperature. Supernatant was decanted and cell pellet was re-suspended in BMM to an OD600 of 1.0 (100-200 ml BMM). The culture was placed in a 1-litter baffled flask and returned to incubator to continue growth at 250-300 rpm at 28-30 C.
[0071] 3. 100% methanol was added to a final concentration of 0.5% to maintain inducted expression.
[0072] 4. After 96 h, the expression culture was centrifuged for 2-3 min, supernatant was transferred to a separate tube and stored at 80 C. for purification of expression product.
[0073] The supernatant of the culture after 96 h induction was analyzed. Total mount of protein was 0.23 mg/ml. The molecular weight of the protein product is consistent with the predicted value of 78 kDa by BioEdit.
[0074] The expression of the recombinant wtyDPP is as follows: the recombinant vector in the expression process of the recombinant myDPP is replaced by the vector pHIL-S1-wtyDPP, and the other operations are the same as that of the recombinant myDPP.
Example 4: Purification of Recombinant myDPP and wtyDPP
[0075] The recombinant expression culture was precipitated with 70% saturation (NH.sub.4).sub.2SO.sub.4, producing crude enzyme as precipitate. The crude enzyme was dissolved in equal volume of PBS, centrifuged. The supernatant was loaded on a hydrophobic Phenyl Sepharose column; active products were collected from gradient elution. The product was subjected to dialysis desalination and concentrated after equilibration with PBS. The active peak was eluted using pH gradient and fraction collected. The PBS solution is concentrated after being balanced. The method specifically comprises the following steps:
[0076] I. Crude Enzyme from (NH.sub.4).sub.2SO.sub.4 Precipitation
[0077] (NH.sub.4).sub.2SO.sub.4 powder was added to the recombinant expression culture until 40% saturation followed by centrifugation at 10000 g for 20 min at 4 C. The supernatant was added more (NH.sub.4).sub.2SO.sub.4 until 70% saturation. Crude enzyme was obtained from centrifugation at 10000 g for 20 min at 4 C.
[0078] II. Hydrophobic Interaction Chromatography
[0079] The crude enzyme was dissolved in equal volume of 0.02 M PBS (pH 6.0). and centrifuged at 4000 g for 10 min at 4 C. Supernatant was loaded on a Phenyl Sepharose column (Pharmacia Biotech. Inc.) which had been washed to background using 0.02M PBS+30% saturation (NH.sub.4).sub.2SO.sub.4, pH 6.0. Gradient elution with solution A (0.02M PBS+10% saturation (NH.sub.4).sub.2SO.sub.4, pH 6.0) and solution B (0.02 M PBS, pH 6.0) gave an active product. The product was subjected to dialysis desalination and concentrated after equilibration with F solution (0.02 M PBS+5 M NaCl, pH 7.5) to 1 mg/ml. The object product peak is identified by SDS-PAGE electrophoresis.
Example 5: Test of the Oxidative Decomposition Activity of Recombinant Proteins myDPP and wtyDPP on 6-methoxy-bifuran coumarin
[0080] The enzyme unit (U) is a unit for the amount of a particular enzyme. One U is defined as the amount of the enzyme that produces 1 mol H.sub.2O.sub.2 that is, the amount that catalyzes the conversion of 1 micro mole of substrate per minute.
[0081] The method of measuring the enzyme activity:
[0082] 30 l of substrate (6-methoxy-bifuran coumarin) with the concentration of 100 g/ml into 10 ml of enzyme with the concentration of 10 g/ml. Let the mixture react for 10 minutes at 25 C. and pH6.5, and then 200 l of Horseradish Peroxidase (HRP) with the concentration of 0.34 mg/ml and 200 l of 3, 3-5, 5-tetramethyl benzidine (TMB) with the concentration of 5 mM were added, and then developing for 30 minutes, then measuring the light absorption value at the ultraviolet of 650 nm, and calculating the enzyme activity unit.
[0083] The enzyme activity result shows that protein wtyDPP has no decomposition activity on 6-methoxy-bifuran coumarin, but the protein myDPP has a decomposition activity on 6-methoxy-bifuran coumarin, and its relative enzyme activity is 33.61 U/mg.
TABLE-US-00001 TABLE 1 Sample Processing Method Reaction System Group Steps of the Method wtyDPP Group myDPP 1. wtypDPP enzyme solution was added. 10 ul 2. myDPP enzyme solution was added. 10 ul 3. 6-methoxy-bifuran coumarin solution was 30 ul 30 ul added. 4. The reaction was carried out at the pH 6.5 and 25 C. for 30 minutes. 5. Horseradish peroxidase was added. 200 ul 200 ul 6. 3,3-5,5-tetramethyl benzidine was added. 200 ul 200 ul 7. Methyl alcohol was added. 200 ul 200 ul 8. The mixture was subjected to color while mixing for 30 minutes, and then the light absorbance value was measured in 650 nm.