High Efficient Expression Of Two Specific Repetitive Sequence Proteins And Their Application In Biochemical Engineering

Collagen like proteins (CLP) and elastin like polypeptides (ELP) contain the highly repetitive amino acid sequences that self-assemble into regular secondary and higher order stuctures to provide special biological functions. They have been widely used in biomedical and tissue engineering. Efficient expression of these proteins with specific repetitive sequences was developed and their applications in many fields were also studied in this study.Firstly, systematic studies were carried out in this study to produce human like collagen (HLC) in E. coli. By co-expressing the HLC gene with human prolyl 4-hydroxylase and D-arabinono-1,4-lactone oxidase in E. coli, the high efficient expression of HLC was achieved. The results showed that the yield of recombinant HLC was 49.55 mg/L in shaking flasks, when cells were grew in MBL medium under the optimization of culture and induction conditions. By adopting the glucose feeding strategy, the expression level of target HLC can be further improved up to 0.26 g/L in a 10-L bench-top fermentor. Further HPLC analyses revealed that more than 10% of proline residues in purified HLC were successfully hydroxylated.Then, systematic studies were carried out to produce another CLP (Scl2 from S. pyogenes) in E. coli. The intergrin binding domain (GERGFPGERGVE), heparin binding sequence (GRPGKPGKQGQK) and RGD was integrated into the protein sequence of Scl2 to create a multifunctional Scl2-M. By adopting the glycerol feeding strategy, the expression level of Scl2-M can be improved up to 15.23 g/L in a 10-L bench-top fermentor. By using Ni2+ affinity chromatograph, the purified Scl2-M protein showed a purity of 94.5% and the recovery was about 73.1%. In addition, the experiment showed that recombinant protein could effectively promote the proliferation of THP-1.The subsequent separation and purification of human basic fibroblast growth factor (hbFGF) by using Scl2-M as fusion protein was also established. By adopting the glycerol feeding strategy, the expression level of fusion protein can be improved up to 11.32 g/L in a 10-L bench-top fermentor and the yield of hbFGF was about 3.83 g/L. The purified hbFGF and Scl2-M were obtained by using Ni2+ affinity chromatography, enzyme digestiton and CM-sepharose ion exchange chromatography. The total recovery of hbFGF and Scl2-M was about 38.3% and 53.4%, respectively. The result also indicated that 20 ng/mL of hbFGF can effectively promote the proliferation of fibroblast NIH3T3 by the MTT method.Finally, systematic investigation was carried out to use the ELP tag to purify the L-glutamate oxidase (LGOX). Batch and fed batch cultivation of the recombinant cells were carried out in a 30 L fermentor with glycerol as substrate. By adopting the glycerol feeding strategy, the expression level of target protein can be improved up to 74.25 U/mL in the fermentor. Then, mature LGOX was purified by inverse transition cycling and proteinase K digestion, the relative enzyme activity was 98.14 U/mg and the recovery rate was 64.4%.The obtained LGOX was then used to produce a-ketoglutaric acid (α-KG). Firstly, the catalytic reaction conditions of free enzyme and immobilized enzyme were optimized in a 3 L reactor. By the addition of substrate and catalase,142.5 g/L a-KG mixed with 4.23 g/L succinate was obtained in the 3 L reactor. Repeated catalysis study by using the immobilized enzyme was also carried out and this immobilized enzyme can be used for 25 times by repeated batch production of a-KG in an 800 L reactor. The average producing rate of a-KG was about 6.08 g/L/h and the average conversion rate of L-glutamate reached about 94.5%.Through our work, we have successfully assembled post-translational machinery for proline hydroxylation of recombinant HLC in a simple prokaryotic system. The modified Scl2-M was also studied, and then it was used as the fusion protein tag to prepare hbFGF. The mature LGOX was prepared by using the ELP tag and the digestion of proteinase K. By using this enzyme, the high value-added of a-KG was obtained from L-glutamate.