Enhancing recombinant protein yields in Escherichia coli using the T7 system under the control of heat inducible λPL promoter

被引:23
作者
Gupta, JC [1 ]
Jaisani, M [1 ]
Pandey, G [1 ]
Mukherjee, KJ [1 ]
机构
[1] Jawaharlal Nehru Univ, Ctr Biotechnol, New Delhi 110067, India
关键词
recombinant; E-coli; overexpression; beta-galactosidase; T7; promoter; lambda P-L promoter; heat shock; fed batch;
D O I
10.1016/S0168-1656(98)00193-X
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A recombinant plasmid containing the complete lacZ gene downstream of the T7 promoter was used to transform Escherichia coli containing another plasmid which had the T7 RNA polymerase gene under the control of heat inducible lambda P-L promoter. This recombinant E. coli containing the two plasmids was studied in order to enhance beta-galactosidase expression. The hear shock time which effectively regulates the T7 RNA polymerase was optimized and best expression of beta-galactosidase was obtained with 2 min heat shock. Substrate feeding increased the duration of log phase and allowed induction at a higher cell density without affecting the specific activity. A high cell density (7 g l(-1)) and high specific activity (similar to 20 000 U) were achieved which effectively increased the product concentration 18-fold. (C) 1999 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:125 / 134
页数:10
相关论文
共 22 条
[1]   NEW RNA POLYMERASE FROM ESCHERICHIA-COLI INFECTED WITH BACTERIOPHAGE-T7 [J].
CHAMBERL.M ;
MCGRATH, J ;
WASKELL, L .
NATURE, 1970, 228 (5268) :227-&
[2]   FEEDBACK-REGULATION OF RIBOSOMAL-RNA AND TRANSFER-RNA SYNTHESIS AND ACCUMULATION OF FREE RIBOSOMES AFTER CONDITIONAL EXPRESSION OF RIBOSOMAL-RNA GENES [J].
GOURSE, RL ;
TAKEBE, Y ;
SHARROCK, RA ;
NOMURA, M .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1985, 82 (04) :1069-1073
[3]  
HUI A, 1987, METHOD ENZYMOL, V153, P432
[4]   PRODUCTION OF RECOMBINANT HUMAN GROWTH-HORMONE IN ESCHERICHIA-COLI - EXPRESSION OF DIFFERENT PRECURSORS AND PHYSIOLOGICAL-EFFECTS OF GLUCOSE, ACETATE, AND SALTS [J].
JENSEN, EB ;
CARLSEN, S .
BIOTECHNOLOGY AND BIOENGINEERING, 1990, 36 (01) :1-11
[5]   THE EFFECTS OF PLASMID CONTENT, TRANSCRIPTION EFFICIENCY, AND TRANSLATION EFFICIENCY ON THE PRODUCTIVITY OF A CLONED GENE PROTEIN IN ESCHERICHIA-COLI [J].
KIM, JY ;
RYU, DDY .
BIOTECHNOLOGY AND BIOENGINEERING, 1991, 38 (11) :1271-1279
[6]   Effects of glycerol on beta-lactamase production during high cell density cultivation of recombinant Escherichia coli [J].
Kwon, S ;
Kim, S ;
Kim, E .
BIOTECHNOLOGY PROGRESS, 1996, 12 (02) :205-208
[7]   MATHEMATICAL-MODELING OF INDUCED FOREIGN PROTEIN-PRODUCTION BY RECOMBINANT BACTERIA [J].
LEE, J ;
RAMIREZ, WF .
BIOTECHNOLOGY AND BIOENGINEERING, 1992, 39 (06) :635-646
[8]  
MILLER JM, 1972, MOL GENET, P352
[9]   EFFECT OF INDUCTION TEMPERATURE ON THE PRODUCTION OF MALARIA ANTIGENS IN RECOMBINANT ESCHERICHIA-COLI [J].
OKITA, B ;
ARCURI, E ;
TURNER, K ;
SHARR, D ;
DELTITO, B ;
SWANSON, J ;
SHATZMAN, A ;
ZABRISKIE, D .
BIOTECHNOLOGY AND BIOENGINEERING, 1989, 34 (06) :854-862
[10]   PLASMID VECTORS FOR HIGH-EFFICIENCY EXPRESSION CONTROLLED BY THE PL PROMOTER OF COLIPHAGE-LAMBDA [J].
REMAUT, E ;
STANSSENS, P ;
FIERS, W .
GENE, 1981, 15 (01) :81-93