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DsbA 1

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chaperon 1

soluble expression 1

trypsin 1

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DsbA-DsbA

Ye Liu,Wenyong Zhang,Xubin Yang,Guangbo Kang,Damei Wang,He Huang

Frontiers of Chemical Science and Engineering 2015, Volume 9, Issue 4,   Pages 511-521 doi: 10.1007/s11705-015-1519-1

Abstract: A co-expressing system of DsbA-DsbA 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-DsbA-DsbA -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 BL21 (DE3) strain with vector pET28a-DsbA-DsbA -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 -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 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 -trypsin compared with DsbA-trypsin, DsbA may function as charge or hydrophobic balance in recombinant protein DsbA-DsbA -trypsin.

Keywords： DsbA     DsbA-DsbAmut     soluble expression     trypsin     chaperon    

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DsbA-DsbA

Ye Liu,Wenyong Zhang,Xubin Yang,Guangbo Kang,Damei Wang,He Huang

Journal Article