Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia Coli

被引:136
作者
Nallamsetty, S [1 ]
Austin, BP [1 ]
Penrose, KJ [1 ]
Waugh, DS [1 ]
机构
[1] NCI, Macromol Crystallog Lab, Canc Res Ctr, Frederick, MD 21702 USA
关键词
immobilized metal affinity chromatography; IMAC; maltose-binding protein; Gateway cloning; fusion tag; structural genomics; TEV protease; expression vector; fusion protein;
D O I
10.1110/ps.051718605
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
Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His(6)MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His(6)MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.
引用
收藏
页码:2964 / 2971
页数:8
相关论文
共 27 条
[1]   VECTORS BEARING A HYBRID TRP-LAC PROMOTER USEFUL FOR REGULATED EXPRESSION OF CLONED GENES IN ESCHERICHIA-COLI [J].
AMANN, E ;
BROSIUS, J ;
PTASHNE, M .
GENE, 1983, 25 (2-3) :167-178
[2]   Escherichia coli maltose-binding protein as a molecular chaperone for recombinant intracellular cytoplasmic single-chain antibodies [J].
Bach, H ;
Mazor, Y ;
Shaky, S ;
Shoham-Lev, A ;
Berdichevsky, Y ;
Gutnick, DL ;
Benhar, I .
JOURNAL OF MOLECULAR BIOLOGY, 2001, 312 (01) :79-93
[3]   Turning protein crystallisation from an art into a science [J].
Chayen, NE .
CURRENT OPINION IN STRUCTURAL BIOLOGY, 2004, 14 (05) :577-583
[4]   ENGINEERING RIBONUCLEASE-A - PRODUCTION, PURIFICATION AND CHARACTERIZATION OF WILD-TYPE ENZYME AND MUTANTS AT GLN11 [J].
DELCARDAYRE, SB ;
RIBO, M ;
YOKEL, EM ;
QUIRK, DJ ;
RUTTER, WJ ;
RAINES, RT .
PROTEIN ENGINEERING, 1995, 8 (03) :261-273
[5]   Three-dimensional structure of the type III secretion chaperone SycE from Yersinia pestis [J].
Evdokimov, AG ;
Tropea, JE ;
Routzahn, KM ;
Waugh, DS .
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, 2002, 58 :398-406
[6]   Maltodextrin-binding proteins from diverse bacteria and archaea are potent solubility enhancers [J].
Fox, JD ;
Routzahn, KM ;
Bucher, MH ;
Waugh, DS .
FEBS LETTERS, 2003, 537 (1-3) :53-57
[7]  
Fox Jeffrey D, 2003, Methods Mol Biol, V205, P99
[8]   Expression of an active recombinant lysine 49 phospholipase A2 myotoxin as a fusion protein in bacteria [J].
Giuliani, CD ;
Iemma, MRC ;
Bondioli, ACV ;
Souza, DHF ;
Ferreira, LL ;
Amaral, AC ;
Salvini, TF ;
Selistre-de-Araujo, HS .
TOXICON, 2001, 39 (10) :1595-1600
[9]   Soluble expression of a functionally active Plasmodium falciparum falcipain-2 fused to maltose-binding protein in Escherichia coli [J].
Goh, LL ;
Loke, P ;
Singh, M ;
Sim, TS .
PROTEIN EXPRESSION AND PURIFICATION, 2003, 32 (02) :194-201
[10]   Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli [J].
Hammarström, M ;
Hellgren, N ;
Van den Berg, S ;
Berglund, H ;
Härd, T .
PROTEIN SCIENCE, 2002, 11 (02) :313-321