The solubility and stability of recombinant proteins are increased by their fusion to NusA

被引：132

作者：

De Marco, V ^{[1
]}

Stier, G ^{[1
]}

Blandin, S ^{[1
]}

de Marco, A ^{[1
]}

机构：

[1] EMBL Heidelberg, D-69117 Heidelberg, Germany

来源：

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS | 2004年 / 322卷 / 03期

关键词：

expression vector; GST; NusA; recombinant protein; Escherichia coli; protein degradation;

D O I：

10.1016/j.bbrc.2004.07.189

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20 mg of fusion protein per litre of culture and 2.5-5 mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins. (C) 2004 Elsevier Inc. All rights reserved.

引用

页码：766 / 771

页数：6

共 23 条

[1] A set of pBR322-compatible plasmids allowing the testing of chaperone-assisted folding of proteins overexpressed in Escherichia coli
Castanie, HP
Berges, H
Oreglia, J
Prere, MF
Fayet, O
[J]. ANALYTICAL BIOCHEMISTRY, 1997, 254 (01) : 150 - 152
[2] PRODUCTION OF RECOMBINANT BOVINE ENTEROKINASE CATALYTIC SUBUNIT IN ESCHERICHIA-COLI USING THE NOVEL SECRETORY FUSION PARTNER DSBA
COLLINSRACIE, LA
MCCOLGAN, JM
GRANT, KL
DIBLASIOSMITH, EA
MCCOY, JM
LAVALLIE, ER
[J]. BIO-TECHNOLOGY, 1995, 13 (09): : 982 - 987
[3] Davis GD, 1999, BIOTECHNOL BIOENG, V65, P382, DOI 10.1002/(SICI)1097-0290(19991120)65:4<382::AID-BIT2>3.3.CO
[4] 2-9
[5] Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning
de Marco, A
De Marco, V
[J]. JOURNAL OF BIOTECHNOLOGY, 2004, 109 (1-2) : 45 - 52
[6] Recombinant maize protoporphyrinogen IX oxidase expressed in Escherichia coli forms complexes with GroEL and DnaK chaperones
de Marco, A
Volrath, S
Bruyere, T
Law, M
Fonné-Pfister, R
[J]. PROTEIN EXPRESSION AND PURIFICATION, 2000, 20 (01) : 81 - 86
[7] Dimerization properties of a Xenopus laevis kinesin-II carboxy-terminal stalk fragment
De Marco, V
de Marco, A
Goldie, KN
Correia, JJ
Hoenger, A
[J]. EMBO REPORTS, 2003, 4 (07) : 717 - 722
[8] Ford C F, 1991, Protein Expr Purif, V2, P95, DOI 10.1016/1046-5928(91)90057-P
[9] Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused
Kapust, RB
Waugh, DS
[J]. PROTEIN SCIENCE, 1999, 8 (08) : 1668 - 1674
[10] A THIOREDOXIN GENE FUSION EXPRESSION SYSTEM THAT CIRCUMVENTS INCLUSION BODY FORMATION IN THE ESCHERICHIA-COLI CYTOPLASM
LAVALLIE, ER
DIBLASIO, EA
KOVACIC, S
GRANT, KL
SCHENDEL, PF
MCCOY, JM
[J]. BIO-TECHNOLOGY, 1993, 11 (02): : 187 - 193

← 1 2 3 →