BIOCATALYST AS A CORE COMPONENT OF AN ENZYME-CATALYZED REDOX SYSTEM FOR THE BIOCATALYTIC REDUCTION OF CYSTINE

Abstract

An enzyme for reducing cystine to cysteine is a fusion protein that includes the protein activities of thioredoxin (protein i) having KEGG database number EC 1.8.4.8 or EC 1.8.4.10 and thioredoxin reductase (protein ii) having KEGG database number EC 1.8.1.9. The thioredoxin (protein i) is the protein activity of thioredoxin 1 from E. coli and the thioredoxin reductase (protein ii) is the protein activity of the thioredoxin reductase from E. coli. The activity of the fusion protein is at least 100% of the activity of a mixture of the same but unfused individual proteins i and ii. The fusion protein has the enzyme activity to reduce cystine to cysteine. The coding sequences (cds) responsible for the activity of protein i and ii has been fused.

Claims

1-15. (canceled)

16. An enzyme for reducing cystine to cysteine, wherein the enzyme is a fusion protein that comprises the protein activities of i) thioredoxin (protein i) having KEGG database number EC 1.8.4.8 or EC 1.8.4.10, wherein thioredoxin (protein i) is the protein activity of thioredoxin 1 from E. coli, and ii) thioredoxin reductase (protein ii) having KEGG database number EC 1.8.1.9, wherein the thioredoxin reductase (protein ii) is the protein activity of the thioredoxin reductase from E. coli, wherein the activity of the fusion protein is at least 100% of the activity of a mixture of the same but unfused individual proteins i and ii, wherein the fusion protein has the enzyme activity to reduce cystine to cysteine and wherein the coding sequences (cds) responsible for the activity of protein i and ii have been fused.

17. The enzyme as claimed in claim 16, wherein the fusion protein comprises two amino acid sequences, wherein one of these amino acid sequences is at least 50% identical to SEQ ID No. 7, the other amino acid sequence is at least 50% identical to SEQ ID No. 8 and the fusion protein has CR activity.

18. The enzyme as claimed in claim 16, wherein the amino acid sequence of the protein located N-terminally is in the fusion protein shortened C-terminally by one to at most five amino acids.

19. The enzyme as claimed in claim 16, wherein the amino acid sequence of the protein located C-terminally is in the fusion protein shortened N-terminally by one to at most five amino acids.

20. The enzyme as claimed in claim 16, wherein the amino acid sequence of the fused protein activities of thioredoxin (protein i) and thioredoxin reductase (protein ii) are in the fusion protein connected by a linker sequence of one to at most five amino acids.

21. The enzyme as claimed in claim 16, wherein the fusion protein is an amino acid sequence selected from the group consisting of SEQ ID No. 9, SEQ ID No. 10, and SEQ ID No. 28.

22. A process for enzymatically reducing cystine to cysteine, wherein cystine is reduced by an enzyme as claimed in claim 16 in the presence of a cofactor, wherein two molecules of L-cysteine are formed from one molecule of the chemical compound cystine.

23. The process as claimed in claim 22, wherein the reduction takes place at a pH of from 6 to 9.

24. The process as claimed in claim 22, wherein the cofactor is a substance selected from the group consisting of NADPH and NADH.

25. The process as claimed in claim 22, wherein the process includes a cofactor-regenerating enzyme.

26. The process as claimed in claim 25, wherein the cofactor-regenerating enzyme is a dehydrogenase, with the reduction additionally taking place in the presence of an electron donor.

27. The process as claimed in claim 26, wherein the dehydrogenase is the alcohol dehydrogenase, with isopropanol used as the electron donor.

Description

EXAMPLES

Example 1: Generation of the Cystine Reductase System TrxA, TrxB, TrxBA, and TrxAB

[0074] Preparation of the Expression Vector:

[0075] As the vector for the expression of the DNA sequences coding for the corresponding candidate proteins TrxA, TrxB, TrxAB, TrxBA, TrxB5A or MI-TrxBA, the expression plasmid pGJ3477 was selected. This is a medium- to high-copy plasmid (50-60 copies per cell) based on the ColE1 origin of replication. The plasmid map is shown in FIG. 1, in which the positions of common, only single-cutting restriction enzymes (with 6-base recognition sequence) are indicated on the plasmid map. The sequence is specified in SEQ ID No. 11.

[0076] In this plasmid, the coding sequence of the respective candidate was placed under the control of the arabinose-inducible promoter P.sub.BAD.

[0077] First, the entire expression plasmid was amplified by inverse PCR: [0078] 50 ng of pGJ3477 DNA, 0.5 pmol of the respective primers 3477-fwrd (SEQ ID No. 12) and 3477-rev (SEQ ID No. 13), Q5® reaction buffer (New England Biolabs, NEB), 1 unit of Q5® DNA polymerase (NEB) in an end volume of 50 μl. [0079] PCR program: 1 min at 98° C., then 30 cycles of 30 s at 98° C., 30 s at 65° C. (annealing), and 2 min at 72° C. (synthesis).

[0080] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mix and the mixture was incubated at 37° C. for 1 h. This was followed by chromatographic purification of the DNA (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0081] Cloning of trxA and trxB:

[0082] For the cloning of the protein-coding sequences trxA (SEQ ID No. 2) and trxB (SEQ ID No. 3), oligonucleotide primers were defined, the target-gene-specific sequences of which were extended by at least 15 nucleotides, which were overlapping with the sequences at the end of the vector DNA. The genes were amplified by PCR from the E. coli genome of BL21 (colony PCR).

[0083] The following mixtures were chosen for amplification by PCR: [0084] 50 ng of genomic E. coli BL21 DNA (NEB), 0.5 pmol of the respective primers trxA-fwrd (SEQ ID No. 14) and trxA-rev (SEQ ID No. 15) or trxB-fwrd (SEQ ID No. 16) and trxB-rev (SEQ ID No. 17), Q5® reaction buffer (NEB), 1 unit of Q5® DNA polymerase (NEB) in an end volume of 50 μl. [0085] PCR program: 1 min at 98° C., then 30 cycles of 30 s at 98° C., 30 s at 60° C. (annealing), and 15 s (trxA) or 30 s (trxB) at 72° C. (synthesis).

[0086] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mix and the mixture was incubated at 37° C. for 1 h. This was followed by chromatographic purification of the DNA (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0087] LIC-PCR:

[0088] In general, the protein-coding DNA sequences were introduced base-exactly into the basic expression vector pGJ3477 via LIC-PCR (ligation-independent-cloning of PCR products) as described in Aslanidis C. and de Jong P. J., Nucleic Acids Res. 18, pp. 6069-6074).

[0089] For this purpose, 50 ng of the purified coding DNA of the respective candidate protein was used in the LIC-PCR reaction together with 50 ng of the prepared vector DNA. The LIC-PCR mixture was then, using standard methods, transformed in E. coli XL1 Blue cells and plated on selective LB medium (LB+100 mg/L ampicillin) and incubated at 37° C. for 18 h. For identification of correct clones, plasmid DNA was isolated from the colonies obtained and the expression cassette was fully sequenced.

[0090] The resulting plasmids, which were composed of the expression vector pGJ3477 and the coding DNA sequence trxA or trxB, were referred to hereinafter as the trxA or trxB expression vector.

[0091] Cloning of trxAB and trxBA:

[0092] The cloning of the DNA trxAB or trxBA respectively encoding the corresponding fusion protein TrxAB or TrxBA was carried out in analogous manner to the cloning of trxA and trxB as described above, by inserting the trxA sequence into the trxB expression vector by LIC-PCR between the N-terminal his-tag and the sequence trxB encoding the protein TrxB or by inserting the trxB sequence into the trxA expression vector by LIC-PCR between the his-tag and the sequence trxA encoding the protein TrxA: The following mixtures were chosen for amplification by PCR: [0093] The vector DNA (trxA or trxB expression vector) was amplified by inverse PCR with the primers vtrxA-fwrd (SEQ ID No. 18) and 3477-rev (SEQ ID No. 13) or vtrxB-fwrd (SEQ ID No. 21) and 3477-rev (SEQ ID No. 13), for which the following PCR program was selected: 2 min at 98° C., then 30 cycles of 45 s at 98° C./30 s at 60° C./2.5 min at 72° C. [0094] The trxA or trxB gene segments for the fusion proteins were amplified by PCR with the primers ftrxA-fwrd (SEQ ID No. 22) and ftrxA-rev (SEQ ID No. 23) or ftrxB-fwrd (SEQ ID No. 19) and ftrxB-rev (SEQ ID No. 20), for which the following PCR program was selected: 2 min at 98° C.->then 30 cycles of 45 s at 98° C./30 s at 60° C./15 s at 72° C. BL21 genomic DNA was used as a template.

[0095] At the end of the PCR reactions, the restriction enzyme DpnI (10 units, NEB) was added to the respective reaction mix and the mixture was incubated at 37° C. for 1 h. This was followed by chromatographic purification of the insert DNA (Macherey & Nagel; NucleoSpin® Gel and PCR Clean-up-Kit).

[0096] The LIC-PCR reaction was carried out using 50 ng of the amplified vector DNA of the trxA or trxB expression vector and 75 ng of the insert DNA (trxB or trxA). The LIC-PCR mixture was then, using standard methods, transformed in E. coli XL1 Blue cells and plated on selective LB medium (LB+100 mg/L ampicillin) and incubated at 37° C. for 18 h. For identification of correct clones, plasmid DNA was isolated from the colonies obtained and the expression cassette was fully sequenced. SEQ ID No. 9 and SEQ ID No. 10 specify the amino acid sequences of the fusion proteins TrxBA and TrxAB resulting respectively from the expression of the sequences SEQ ID No. 4 and SEQ ID No. 5.

Example 2: Cloning of Cystine Reductase MI-TrxBA

[0097] As described in the introduction, Mycobacterium leprae possesses a gene segment that encodes for the protein with homology to thioredoxin (Trx) and thioredoxin reductase (TR) (Wieles B. et al. 1995, J. Biol. Chem. 270, pp. 25604-25606). The sequence can be taken from the public databases (NCBI Reference Sequence: WP_010909042.1).

[0098] The sequence taken from the public databases was customized in silico to the codon usage of the host E. coli. This was done using the IDT web server (www.idtdna.com). For cloning into the target vector pGJ3477, the coding region was extended at the 5′ and 3′ ends by sequences that overlap with the vector sequence (see also cloning of trxA and trxB). The resulting total DNA sequence was produced synthetically (specified in SEQ ID No. 24) by Geneart (www.thermofisher.com) and designated MI-trxBA (also referred to as ml-trxBA).

[0099] The synthetic MI-trxBA sequence was cloned into the vector pGJ3477 in analogous manner to the cloning of the trxA and trxB expression vectors by LIC-PCR. For the cloning of the protein-coding sequence MI-trxBA (SEQ ID No. 24), oligonucleotide primers were defined, the target-gene-specific sequences of which were extended by at least 15 nucleotides, which were overlapping with the sequences at the end of the vector DNA. The synthetic gene served as a template.

[0100] The following mixtures were chosen for amplification by PCR: [0101] 20 ng of the template (Geneart), 0.5 pmol of the respective primers MI-trxBA-fwrd (SEQ ID No. 25) and MI-trxBA-rev (SEQ ID No. 26), Q5® reaction buffer, 1 unit of Q5® DNA polymerase (NEB) in an end volume of 50 μl. [0102] PCR program: 1 min at 98° C., then 30 cycles of 30 s at 98° C., 30 s at 60° C. (annealing), and 60 s at 72° C. (synthesis).

[0103] At the end of the reaction, the restriction enzyme DpnI (10 units, NEB) was added to the reaction mix and the mixture was incubated at 37° C. for 1 h. This was followed by chromatographic purification of the DNA (Macherey & Nagel: NucleoSpin® Gel and PCR Clean-up-Kit).

[0104] LIC-PCR:

[0105] The protein-coding DNA sequence was introduced base-exactly into the basic expression vector pGJ3477 via LIC-PCR (ligation-independent-cloning of PCR products) as described in Aslanidis C. and de Jong P. J., Nucleic Acids Res. 18, pp. 6069-6074).

[0106] For this purpose, 50 ng of the purified coding DNA was used in the LIC-PCR reaction together with 60 ng of the prepared vector DNA.

[0107] The LIC-PCR mixture was then, using standard methods, transformed in E. coli XL1 Blue cells and plated on selective LB medium (LB+100 mg/L ampicillin) and incubated at 37° C. for 18 h. For identification of correct clones, plasmid DNA was isolated from the colonies obtained and the expression cassette was fully sequenced. The resulting plasmid, which was composed of the expression vector pGJ3477 and the coding DNA sequence MI-trxBA, was referred to hereinafter as the MI-trxBA expression vector.

Example 3: Cloning of Cystine Reductase TrxB5A (with Linker Sequence)

[0108] Cloning of trxB5A:

[0109] The cloning of the DNA trxB5A encoding the corresponding fusion protein TrxB5A was carried out in analogous manner to the cloning of trxBA as described above, by inserting a TrxB sequence extended C-terminally by 5 amino acids (referred to hereinafter as TrxB5, and the cds as trxB5) into the trxA expression vector by LIC-PCR between the his-tag and the sequence trxA encoding the protein TrxA:

[0110] The following mixtures were chosen for amplification by PCR: [0111] The vector DNA (trxA expression vector) was amplified by inverse PCR with the primers vtrxA5-fwrd (SEQ ID No. 29) and 3477-rev (SEQ ID No. 13), for which the following PCR program was selected: 2 min at 98° C., then 30 cycles of 45 s at 98° C./30 s at 60° C./2.5 min at 72° C. [0112] trxB5 was amplified by PCR with the primers ftrxB-fwrd (SEQ ID No. 19) and ftrxB5-rev (SEQ ID No. 30), for which the following PCR program was selected: 2 min at 98° C.->then 30 cycles of 45 s at 98° C./30 s at 60° C./15 s at 72° C. BL21 genomic DNA was used as a template.

[0113] At the end of the PCR reactions, the restriction enzyme DpnI (10 units, NEB) was added to the respective reaction mix and the mixture was incubated at 37° C. for 1 h. This was followed by chromatographic purification of the insert DNA (Macherey & Nagel; NucleoSpin® Gel and PCR Clean-up-Kit).

[0114] The LIC-PCR reaction was carried out using 50 ng of the amplified vector DNA of the trxA expression vector and 75 ng of the insert DNA (trxB5).

[0115] The LIC-PCR mixture was then, using standard methods, transformed in E. coli XL1 Blue cells and plated on selective LB medium (LB+100 mg/L ampicillin) and incubated at 37° C. for 18 h. For identification of correct clones, plasmid DNA was isolated from the colonies obtained and the expression cassette was fully sequenced.

[0116] SEQ ID No. 28 specifies the amino acid sequence of the fusion protein TrxB5A resulting from the expression of the sequence SEQ ID No. 27.

Example 4: Enzyme Activity of the Cystine Reductases

[0117] For the recombinant expression of the proteins TrxA, TrxB, TrxBA, TrxB5A, TrxAB, and MI-TrxBA, the investigated expression plasmids encoding the corresponding protein were introduced into the strain E. coli TOP10 (Thermo Fisher Scientific, MA/USA). The bacterial cells were plated on LB medium containing 100 mg/L ampicillin. 25 ml of LB medium containing 100 mg/L ampicillin and 0.2% arabinose (w/v) was then inoculated with a single colony and incubated at 28° C. in a culture shaker cabinet for 18 h.

[0118] For the isolation of the expressed proteins, the cells were sedimented by centrifugation (10 min at 4000 g), the media supernatant was removed, and the biomass resuspended in lysis buffer (PBS+10% BugBuster (Sigma)). The cells were lysed completely in 15 min at RT as per the manufacturer's instructions in the manual of the BugBuster kit (Sigma) and then insoluble matter was removed by centrifugation (20 min at 9500 rpm as per the manufacturer's instructions). For purification of the supernatant (=cell lysate) by affinity chromatography, the cell lysate obtained was loaded into PBS-equilibrated Protino IDA2000 columns (Macherey & Nagel). After a wash step (7 ml of PBS), the samples were eluted with 5 ml of elution buffer (PBS+200 mM imidazole). The resulting protein concentrations were determined by a Bradford assay (Thermo Fisher).

[0119] The cystine reductase activity of the various enzymes TrxA, TrxB, TrxBA, TrxB5A, TrxAB or MI-TrxBA was determined using the two analytical methods described in detail below. [0120] 1. The first method consisted of the photometric detection method, which is based on the consumption of NADPH and the resulting fall in the absorbance values at 340 nm during the reduction reaction. As shown schematically in FIG. 2, the enzymatic conversion of cystine in the reduction to cysteine is accompanied by the consumption of the cofactor NADPH. [0121] The following assay mixture was used for the photometric determination, the end volume of the samples being 1 ml: [0122] 100 mM phosphate buffer pH 7.4 [0123] 2 mM EDTA [0124] 0.2 mM NADPH (Sigma) [0125] 1 mM cystine (Wacker Chemie AG) [0126] After mixing all assay components except for the enzyme, the reaction was started by adding the enzyme: [0127] 10 μg of enzyme TrxA, TrxB, a mixture of TrxA and TrxB, TrxBA, TrxB5A, TrxAB or MI-TrxBA. The assay mixtures were incubated at room temperature (approx. 25° C.) until the measurement described below. [0128] The NADPH concentration of the various 1 ml assay mixtures was measured at various times using a spectrophotometer (Evolution 201 UV-vis spectrophotometer, Thermo Fisher Scientific) in the form of absorbance values at 340 nm. The absorbance values at 340 nm were used to calculate the enzyme activity of the various samples using the Thermo INSIGHT software from the manufacturer Thermo Fisher Scientific. Analyzed in parallel in each case as negative controls were samples that contained either heat-inactivated enzyme, no enzyme, no cystine as substrate, or no NADPH as cofactor. [0129] The enzyme activity was determined from the linear slope at the start of the recorded curve, with the change (decrease) in absorbance over time indicating the rate of NADPH consumption. Since the same amount of enzyme is always used, the rates can be compared. [0130] 2. In the second method, the cysteine formed from cystine by the cystine reductase activity was detected directly via the free SH group by reaction with the compound DTNB (5,5′-dithiobis-2-nitrobenzoic acid; Ellman's reagent), as shown schematically in FIG. 3, also referred to in the context of the present invention as the DTNB assay. [0131] The following assay mixture was used for the photometric assay, the end volume of the samples being 1 ml: [0132] 100 mM phosphate buffer pH 7.4 [0133] 2 mM EDTA [0134] 0.2 mM NADPH (Sigma) [0135] 1 mM cystine (Wacker Chemie AG) [0136] After mixing all assay components apart from the enzyme, the reaction was started by adding the enzyme: [0137] 10 μg of enzyme TrxA, TrxB, a mixture of TrxA and TrxB, TrxBA, TrxB5A, TrxAB or MI-TrxBA. The assay mixtures were incubated at room temperature (approx. 25° C.) until the measurement described below. [0138] L-Cysteine was quantified by the test described by Lee S.-H. et al. 1995 (Biochemical and Biophysical Research Communications 213, pp. 837-844) using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). Quantification of Cys-TNB was by measurement of the DTNB-mediated absorbance at 412 nm. Analysis by HPLC is also possible.

[0139] Analyzed in parallel in each case as negative controls were samples that contained either heat-inactivated enzyme, no enzyme, no cystine as substrate, or no NADPH as cofactor.

[0140] FIG. 4 shows the relative cystine reductase activity of the enzymes TrxBA, TrxB5A, TrxAB, and MI-Trx and of a (9:1) mixture of TrxA and TrxB according to photometric determination as described above under point 1, the activity of the (9:1) mixture of TrxA and TrxB being normalized to 1 and all other activities set in relation thereto. The (9:1) mixture of TrxA and TrxB is an estimated mixing ratio of the individual enzymes that comparative tests have shown to be the most active mixing ratio.

[0141] FIG. 5 shows the detection of cysteine formation by TrxBA and TrxAB according to the DTNB assay and HPLC as described above under point 2.

[0142] The use of clones that contained a longer linker sequence than TrxB5A in the region of 60 nucleotides between the cds for TrxA and TrxB astonishingly led to significantly lower activity in the fusion protein.

Example 5: Activity of the Enzyme Combination Cystine Reductase and ADH

[0143] A comparison of the enzyme specificity toward the cofactors NADPH and NADP.sup.+ found the cystine reductases TrxBA and TrxAB to be highly specific for the cofactor NADPH. In the tests, no enzyme activity toward cystine was detected with NADP.sup.+. Combination experiments investigated the extent to which the enzyme alcohol dehydrogenase (ADH) is able to convert NADP.sup.+ back into NADPH for the reaction. The following conditions were used for this purpose: [0144] 100 mM phosphate buffer pH 7.4 [0145] 5 μl of isopropanol [0146] 2 mM EDTA [0147] 15-50 μM NADPH or NADP.sup.+ [0148] 1 mM cystine [0149] 5 μg of enzyme TrxBA [0150] 50 μl of cell lysate crude extract or, as a negative control, 50 μl of phosphate buffer (Fast Prep digestion of 0.5 ml of cells in 1.5 ml of 4× phosphate buffer pH 7.4. Production of the cells as described in EP 1 832 658 B1)

[0151] The reaction was incubated at 30° C. for 60 min. Samples were taken at 5-minute intervals and the liberated cysteine was derivatized with DTNB as described in WO 2013/000864 A1. Quantification was by measurement of the DTNB-mediated absorbance at 412 nm.

[0152] FIG. 6 shows the result of the measurement of cysteine formation by TrxBA using the DTNB assay in the presence (with) or absence (without) of ADH as a regenerative system.

ABBREVIATIONS USED IN THE FIGURES

[0153] AraC: AraC gene (repressor gene) [0154] pAraC: Promoter of the AraC gene (repressor gene; rev orientation to P.sub.BAD) [0155] pBAD (in the context of the present invention referred to also as P.sub.BAD): Arabinose-inducible promoter for expression (downstream) of inserted target protein sequences [0156] 6HIS: Coding region for His-tag [0157] term: Transcription terminator [0158] Amp: Ampicillin resistance marker [0159] CR: Cystine reductase [0160] rel.: Relative [0161] bps: Base pairs [0162] t: Time