Controlled intracellular processing of fusion proteins by TEV protease

被引:159
作者
Kapust, RB [1 ]
Waugh, DS [1 ]
机构
[1] NCI, Program Struct Biol, Frederick Canc Res & Dev Ctr, Frederick, MD 21702 USA
关键词
D O I
10.1006/prep.2000.1251
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Here we describe a method for controlled intracellular processing (CIP) of fusion proteins by tobacco etch virus (TEV) protease, A fusion protein containing a TEV protease recognition site is expressed in Escherichia coli cells that also contain a TEV protease expression vector. The fusion protein vector is an IPTG-inducible ColE1-type plasmid, such as a T7 or tac promoter vector. In contrast, the TEV protease is produced by a compatible p15A-type vector that is induced by tetracyclines, Not only is the TEV protease regulated independently of the fusion protein, but its expression is highly repressed in the absence of inducer, Certain fusion partners have been shown to enhance the yield and solubility of their passenger proteins. When CIP is used as a purification step, it is possible to take advantage of these characteristics while both eliminating the need for large amounts of pure protease at a later stage and possibly simplifying the purification process. Additionally, we have observed that in some cases the timing of intracellular proteolysis can affect the solubility of the cleaved passenger protein, allowing it to be directed to either the soluble or the insoluble fraction of the crude cell lysate. This method also makes it possible to quickly gauge the efficiency of proteolysis in vivo, before protein purification has begun and in vitro processing is attempted. (C) 2000 Academic Press.
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收藏
页码:312 / 318
页数:7
相关论文
共 35 条
[1]   Recombinant protein expression in Escherichia coli [J].
Baneyx, F .
CURRENT OPINION IN BIOTECHNOLOGY, 1999, 10 (05) :411-421
[2]   An Escherichia coli host strain useful for efficient overproduction of cloned gene products with NaCl as the inducer [J].
Bhandari, P ;
Gowrishankar, J .
JOURNAL OF BACTERIOLOGY, 1997, 179 (13) :4403-4406
[3]   UBIQUITIN FUSION AUGMENTS THE YIELD OF CLONED GENE-PRODUCTS IN ESCHERICHIA-COLI [J].
BUTT, TR ;
JONNALAGADDA, S ;
MONIA, BP ;
STERNBERG, EJ ;
MARSH, JA ;
STADEL, JM ;
ECKER, DJ ;
CROOKE, ST .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1989, 86 (08) :2540-2544
[4]   MECHANISM OF CONTROL OF DNA-REPLICATION AND INCOMPATIBILITY IN COLE1-TYPE PLASMIDS - A REVIEW [J].
DAVISON, J .
GENE, 1984, 28 (01) :1-15
[5]  
DEBOER HA, 1983, P NATL ACAD SCI-BIOL, V80, P21
[6]   STRUCTURAL REQUIREMENTS OF TETRACYCLINE-TET REPRESSOR INTERACTION - DETERMINATION OF EQUILIBRIUM BINDING CONSTANTS FOR TETRACYCLINE ANALOGS WITH THE TET REPRESSOR [J].
DEGENKOLB, J ;
TAKAHASHI, M ;
ELLESTAD, GA ;
HILLEN, W .
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, 1991, 35 (08) :1591-1595
[7]   MOLECULAR GENETIC-ANALYSIS OF A PLANT-VIRUS POLYPROTEIN CLEAVAGE SITE - A MODEL [J].
DOUGHERTY, WG ;
CARY, SM ;
PARKS, TD .
VIROLOGY, 1989, 171 (02) :356-364
[8]   MODIFIED BACTERIOPHAGE-LAMBDA PROMOTER VECTORS FOR OVERPRODUCTION OF PROTEINS IN ESCHERICHIA-COLI [J].
ELVIN, CM ;
THOMPSON, PR ;
ARGALL, ME ;
HENDRY, P ;
STAMFORD, NPJ ;
LILLEY, PE ;
DIXON, NE .
GENE, 1990, 87 (01) :123-126
[9]   Enhancing recombinant protein yields in Escherichia coli using the T7 system under the control of heat inducible λPL promoter [J].
Gupta, JC ;
Jaisani, M ;
Pandey, G ;
Mukherjee, KJ .
JOURNAL OF BIOTECHNOLOGY, 1999, 68 (2-3) :125-134
[10]   TIGHT REGULATION, MODULATION, AND HIGH-LEVEL EXPRESSION BY VECTORS CONTAINING THE ARABINOSE P-BAD PROMOTER [J].
GUZMAN, LM ;
BELIN, D ;
CARSON, MJ ;
BECKWITH, J .
JOURNAL OF BACTERIOLOGY, 1995, 177 (14) :4121-4130