Transglutaminase variants with improved specificity
A technology of transglutaminase peptide and glutaminase peptide, which is applied in the field of novel transglutaminase variants, can solve problems such as low selectivity, and achieve the effect of increasing yield
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Embodiment 1
[0278] Cloning of propeptide-mTGase in the form of GlyPro-TGase and generation of mutants
[0279] TGase from S. rada ATCC27441
[0280] The sequence of the propeptide mTGase from S. rada (propeptide-mTGase is the peptide produced by the DNA encoding the TGase from S. rada in another microorganism, such as Escherichia coli) such as SEQ ID No.3 shown. The propeptide part is amino acid 1-49 of SEQ ID No.3, and the remaining sequence is the mature mTGase shown in SEQ ID No.1. The mature mTGase part (SEQ ID No.1) is 93.4% identical to the corresponding part of mTGase (SEQ ID No.2) from Streptomyces mobara, as figure 1 shown.
[0281] The 3C-protease sequence LEVLFQGP (3C) was cloned between the propeptide domain of S. rada-TGase (aa 1-49 of SEQ ID No. 3) and the mature mTGase domain. The 3C-protease can specifically cleave between Q and G at the LEVLFQGP site, thereby obtaining two additional amino acid residues, Gly-Pro, added to the N-terminus of the mature mTGase (as sh...
Embodiment 2
[0284] Selectivity of TGase mutants with increased N-terminal amino acid residues
[0285] Preparation of GlyPro-mTGase
[0286] At 30°C, culture pET39b_Met-propeptide-(3C)-mTGase / Escherichia coli BL21(DE3) cells in LB medium supplemented with 30 μg / ml kanamycin to an optical density of 0.4, and then induce the cells with 0.1 mM IPTG 4 hours. Cell pellets were harvested by centrifugation.
[0287] The soluble fraction of the cell pellet was extracted and purified by anion exchange, Q-SEPHAROSEHP column to obtain pure propeptide-(3C)-mTGase protein. The protein was then digested with 3C-protease (from poliovirus) at a ratio of 1:100 (w / w) relative to propeptide-(3C)-mTGase protein overnight at 20°C. The digestion mixture was further purified by a cation exchange column, SP SEPHAROSE HP / Source 30S, to obtain the active mTGase identified by the TGase activity assay.
[0288] Preparation of AlaPro-mTGase
[0289] AlaPro-mTGase was prepared in the same manner as GlyPro-m...
Embodiment 3
[0293] Screening assay for highly selective variants --- for assessing N-terminal extra sequence pair Rada sprockets A Kinetic Approach to Effects of mTGase Selectivity on Thyriticum
[0294] Preparation of hGHO40N and hGHO141N
[0295] The hGHQ40N and hGHQ141N mutants of hGH were constructed by site-directed mutagenesis. They have 4 additional amino acid residues at the N-terminus, were expressed in E. coli as MEAE-hGHQ40N and MEAE-hGHQ141N, and purified in the same manner as wild-type recombinant hGH. Briefly, soluble MEAE-hGH mutants were recovered from crude E. coli lysates by Q SEPHAROSEXL chromatography and further purified with phenyl SEPHAROSE FF. The partially purified MEAE-hGH mutant was digested with DAP-1 enzyme and reacted at 42°C for 1 hour to remove the N-terminal MEAE. Finally, hGH mutants were precipitated with 38% cold ethanol, then dissolved with 7M urea, and purified with a Source 30Q column.
[0296] Kinetic response
[0297] Kinetic reactions ...
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