A novel high-cell-density protein expression system based on Ralstonia eutropha

被引:39
作者
Srinivasan, S [1 ]
Barnard, GC [1 ]
Gerngross, TU [1 ]
机构
[1] Dartmouth Coll, Thayer Sch Engn, Lebanon, NH 03766 USA
关键词
D O I
10.1128/AEM.68.12.5925-5932.2002
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
We describe the development of a novel protein expression system based on the industrial fermentation organism Ralstonia eutropha (formerly known as Alcaligenes eutrophus) NCIMB 40124. This new system overcomes some of the shortcomings of traditional Escherichia coli-based protein expression systems, particularly the propensity of such systems to form inclusion bodies during high-level expression. Using a proteomics approach, we identified promoters that can be induced by simple process parameters or medium compositions in high-density cell culture or shake flasks, respectively. By combining newly developed molecular biological tools with a high-cell-density fermentation process, we were able to produce high levels (> 1 g/liter) of soluble, active organophosphohydrolase, a model enzyme prone to inclusion body formation in E. coli.
引用
收藏
页码:5925 / 5932
页数:8
相关论文
共 46 条
[1]  
Åkesson M, 1999, BIOTECHNOL BIOENG, V64, P590, DOI 10.1002/(SICI)1097-0290(19990905)64:5<590::AID-BIT9>3.0.CO
[2]  
2-T
[3]   The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of Gram-negative bacteria [J].
Alexeyev, MF .
BIOTECHNIQUES, 1999, 26 (05) :824-+
[4]   MODELING OF HIGH CELL-DENSITY FED-BATCH CULTIVATION [J].
ANDERSSON, L ;
STRANDBERG, L ;
HAGGSTROM, L ;
ENFORS, SO .
FEMS MICROBIOLOGY REVIEWS, 1994, 14 (01) :39-44
[5]   A promoter-trap vector for clock-controlled genes in the cyanobacterium Synechocystis sp PCC 6803 [J].
Aoki, S ;
Kondo, T ;
Ishiura, M .
JOURNAL OF MICROBIOLOGICAL METHODS, 2002, 49 (03) :265-274
[6]  
CHARLES M, 1994, BIOPROCESS ENG SYSTE, P5
[7]   HIGH-DENSITY ESCHERICHIA-COLI CULTIVATION PROCESS FOR HYPEREXPRESSION OF RECOMBINANT PORCINE GROWTH-HORMONE [J].
CHEN, HC ;
HWANG, CF ;
MOU, DG .
ENZYME AND MICROBIAL TECHNOLOGY, 1992, 14 (04) :321-326
[8]   HIGH CELL-DENSITY CULTURE OF ESCHERICHIA-COLI IN A FED BATCH SYSTEM WITH DISSOLVED-OXYGEN AS SUBSTRATE FEED INDICATOR [J].
CUTAYAR, JM ;
POILLON, D .
BIOTECHNOLOGY LETTERS, 1989, 11 (03) :155-160
[9]   CHARACTERIZATION OF ORGANOPHOSPHORUS HYDROLASES AND THE GENETIC MANIPULATION OF THE PHOSPHOTRIESTERASE FROM PSEUDOMONAS-DIMINUTA [J].
DAVE, KI ;
MILLER, CE ;
WILD, JR .
CHEMICO-BIOLOGICAL INTERACTIONS, 1993, 87 (1-3) :55-68
[10]   BIOPROCESS DEVELOPMENT TO IMPROVE FOREIGN PROTEIN-PRODUCTION FROM RECOMBINANT STREPTOMYCES [J].
DELACRUZ, N ;
PAYNE, GF ;
SMITH, JM ;
COPPELLA, SJ .
BIOTECHNOLOGY PROGRESS, 1992, 8 (04) :307-315