Publication Date:
2015-07-18
Description:
A co-expressing system of DsbA-DsbA mut was suggested for the first time to enhance the soluble expression of human trypsin-1. As a control, leaderless DsbA chaperone was also co-expressed with human trypsin-1. Vectors pET39b-trypsin and pET28a-DsbADsbA mut -trypsin with the above two DsbA fusion tag were constructed. The strain with vector pET39b-trypsin expressed fusion protein DsbA-trypsin in form of inclusion bodies. While in E. coli BL21 (DE3) strain with vector pET28a-DsbA-DsbA mut -trypsin, the soluble expression of trypsin fusion protein was achieved. Under the optimized expression conditions, the soluble fraction accounted for about 49.43% of total DsbA-DsbA mut -trypsin proteins in crude supernatant. The purification yield was 4.15% by nickel chelating chromatography and 3.3 mg activated trypsin with a purity of 88.68% was obtained from 1 L LB broth. To detect the possible functions of DsbA series chaperons in trypsin fusion protein, we analyzed the primary three-dimensional structure of fusion proteins, mainly focusing on the compatibleness between trypsin and fusion chaperons. The results suggested that (1) besides the primary function in periplasm, leaderless DsbA or DsbA mut may also act as a signal sequences-like leader targeted to periplasm that partly relieved the pressure from fusion protein overexpression and inclusion body formation, and (2) as there was significant soluble expression of DsbA-DsbA mut -trypsin compared with DsbA-trypsin, DsbA mut may function as charge or hydrophobic balance in recombinant protein DsbA-DsbA mut -trypsin.
Print ISSN:
2095-0179
Electronic ISSN:
2095-0187
Topics:
Chemistry and Pharmacology
,
Process Engineering, Biotechnology, Nutrition Technology
