DsbA-DsbA<sup>mut</sup> fusion chaperon improved soluble expression of human trypsinogen-1 in <i>Escherichia coli</i>

doi:10.1007/s11705-015-1519-1

Front. Chem. Sci. Eng.

2015, Vol. 9

Issue (4) : 511-521 https://doi.org/10.1007/s11705-015-1519-1

RESEARCH ARTICLE

DsbA-DsbA^mut fusion chaperon improved soluble expression of human trypsinogen-1 in Escherichia coli

Ye Liu^1,²,Wenyong Zhang^1,^2,⁵,Xubin Yang^1,²,Guangbo Kang³,Damei Wang⁴,He Huang^1,^2,^*(

)

1. Key Laboratory of System Bioengineering, Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
2. Collaborative Innovation Center of Chemical Science and Engineering, Tianjin 300072, China
3. College of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300072, China
4. Gan & Lee Pharmaceuticals, Beijing 101100, China
5. Taiyuan Institute of Technology, Taiyuan 030000, China

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Abstract

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-DsbA-DsbA^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^mutmay 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^mutmay function as charge or hydrophobic balance in recombinant protein DsbA-DsbA^mut-trypsin.

Keywords DsbA DsbA-DsbA^mut soluble expression trypsin chaperon

Corresponding Author(s): He Huang

Online First Date: 20 July 2015 Issue Date: 26 November 2015

Cite this article:

Ye Liu,Wenyong Zhang,Xubin Yang, et al. DsbA-DsbA^mut fusion chaperon improved soluble expression of human trypsinogen-1 in Escherichia coli[J]. Front. Chem. Sci. Eng., 2015, 9(4): 511-521.

URL:

https://academic.hep.com.cn/fcse/EN/10.1007/s11705-015-1519-1
https://academic.hep.com.cn/fcse/EN/Y2015/V9/I4/511

Tab.1 Sequences of primers used in this study

Fig.1 Construction for pET28a-DsbA-DsbA^mut-trypsin expression vector: (a) Construction details for fragment linker-DsbA^mut-linker-DDDDK-MCS (MCS includes ScaI, HindIII, Not1, XhoI restriction sites); (b) Process for construction of pET28a-DsbA-DsbA^mut-trypsin; (c) The expression cassette harbored by pET28a-DsbA-DsbA^mut-trypsin

Fig.2 (a) SDS-PAGE analysis of DsbA-trypsin: lane 1, supernatant (IPTG-induced); lane 2, precipitate (IPTG-induced); lane 3, supernatant (NO IPTG-induced); lane 4, precipitate (NO IPTG-induced). (b) SDS-PAGE analysis of DsbA-trypsin after renaturation: lanes 1~2, supernatant; lane 2, precipitate; lane 3, renaturation reagent control; lane 4, thrombin control; lane 5, porcine trypsin control; lanes 6?7, digestion product by thrombin after 5 h and 12 h, respectively. (c) Expression and detection of DsbA-DsbA^mut-trypsin: lane 1, supernatant (IPTG-induced); lane 2, supernatant (NO IPTG-induced); lane 3, precipitate (IPTG-induced); lane 4, precipitate (NO IPTG-induced)

Fig.3 Disulfide bridges distribution in human trypsin-1. Four disulfide bridges were exposed in surface of human trypsin-1 (C22-C157, C191-C220, C42-C58, C136-C201), and one was buried inherently (C168-C182). The red, blue and azure balls represent positive, negative and nonpolar residues on protein surface, respectively

Fig.4 Purification of fusion protein DsbA-DsbA^mut-trypsin: (a) Imidazole concentration: lane 1, 500 mmol·L⁻¹; lane 2, 50 mmol·L⁻¹; lane 3, 40 mmol·L⁻¹; lane 4, 20 mmol·L⁻¹; lane 5, supernatant (IPTG-induced); (b) SDS-PAGE analysis of the purified fractions: lane 1, supernatant (IPTG-induced); lane 2, precipitate (IPTG-induced). Imidazole concentration in elution buffer: lane 3, 30 mmol·L⁻¹; lane 4, 500 mmol·L⁻¹

Fig.5 Electrostatic potential file of (a) DsbA-linker (reduced type), (b) DsbA-linker (oxidized type), (c) DsbA^mut-linker, and (d) human trypsin-1. The connection amino?acids that distinguishes different parts of DsbA-DsbA^mut-trypsin have been labeled (connections: a, ALA195-c: ALA1; b, ALA195-c: ALA1; c, TYR202-d: ILE16)

Fig.6 Statical hydrophobic patches distribution on protein surface: (a) DsbA-linker (reduced type), (b) DsbA-linker (oxidized type), (c) DsbA^mut-linker, and (d) human trypsin-1. The connection amino acids that distinguishes different parts of DsbA-DsbA^mut-trypsin have been labeled (connections: a, ALA195-c: ALA1, or b, ALA195-c: ALA1; c, TYR202-d: ILE16)

Tab.2 Purification of DsbA-DsbA^mut-trypsin-1 in E. coli

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