大肠杆菌外源蛋白表达载体稳定性的研究进展

摘要/Abstract

摘要： 构建高产、稳定、可靠的质粒载体成为基因重组表达技术的研究重点之一。宿主细胞代谢反应和质粒不稳定性相关信息的缺乏，仍然阻碍着质粒载体的优化，成为限制外源蛋白在大肠杆菌中高效表达的瓶颈之一。主要论述了大肠杆菌外源蛋白表达载体的稳定性，分别从质粒和外源基因的本身特性、重组质粒转化对宿主细胞造成的影响及其他因素等方面阐述了对质粒载体稳定性的影响，同时介绍了相关的解决办法，从而为大肠杆菌表达系统高效表达外源蛋白提供参考。

关键词: 大肠杆菌, 外源蛋白, 表达载体, 稳定性

Abstract: The construction of stable, feasible and high-yielding plasmid vector has become one of research focuses on recombinant technology. The optimization of plasmid vector is still hindered by the lack of information on plasmid instability as well as information on the host metabolic responses. Therefore, this review highlights the stability of plasmid vector when expressing foreign proteins in E. coli and the possible influence factor for its instability, such as the inherent characteristics of the plasmid and the exogenous gene, influence on the host triggered by recombinant plasmid and other factors. Meanwhile, related solutions were briefly introduced, providing a reference for high-level expression of heterologous proteins in E. coli expression system.

Key words: Escherichia, Coli Foreign, Protein Expression vector, Stability

江伟华, 刘益丽, 江明锋. 大肠杆菌外源蛋白表达载体稳定性的研究进展[J]. 生物技术通报, 2014, 0(5): 25-31.

Jiang Weihua, Liu Yili, Jiang Mingfeng. Research Progress on Expression Vector Stability in E. coli System[J]. Biotechnology Bulletin, 2014, 0(5): 25-31.

参考文献

[1] Ertl PF, Thomsen LL. Technical issues in construction of nucleic acid vaccines[J]. Methods, 2003, 31：199-206.
[2] Ribeiro SC, Monteiro GA, Prazeres DM. The impact of polyadenyla-tion signals on plasmid nuclease-resistance and transgene expression[J]. J Gene Med, 2007, 9：392-402.
[3] Hadj Kacem B, Gargouri J, Gargouri A. In vitro direct repeats-mediated deletion during PCR amplification[J]. Mol Biotechnol, 2008, 40：39-45.
[4] Bi X, Liu LF. DNA rearrangement mediated by inverted repeats[J]. Proc Natl Acad Sci USA, 1996, 93：819-823.
[5] Valesova R, Stepanek V, Vecerek B, Kyslik P. IS2-mediated re-arrangement of the promoter sequence suppresses metabolic burden of the recombinant plasmid[J]. Folia Microbiol(Praha), 2005, 50：275-282.
[6] Oliveira PH, Prazeres DM, Monteiro GA. Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress[J]. J Biotechnol, 2009, 143：231-238.
[7] Haddadin FT, Harcum SW. Transcriptome profiles for high-cell-density recombinant and wild-type Escherichia coli[J]. Biotechnol Bioeng, 2005, 90：127-153.
[8] Xu J, Li W, Wu J, et al. Stability of plasmid and expression of a recombinant gonadotropin-releasing hormone(GnRH)vaccine in Escherichia coli[J]. Appl Microbiol Biotechnol, 2006, 73：780-788.
[9] Summers DK, Sherratt DJ. Multimerization of high copy number plasmids causes instability：CoIE1 encodes a determinant essential for plasmid monomerization and stability[J]. Cell, 1984, 36：1097-1103.
[10] Goyal D, Sahni G, Sahoo DK. Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture[J]. Bioresour Technol, 2009, 100：4468-4541.
[11] Krishna Rao DV, Ramu CT, Rao JV, et al. Impact of dissolved oxygen concentration on some key parameters and production of rhG-CSF in batch fermentation[J]. J Ind Microbiol Biotechnol, 2008, 35：991-1000.
[12] Chen HC, Hwang CF, Mou DG. High-density Escherichia coli cultivation process for hyperexpression of recombinant porcine growth hormone[J]. Enzyme Microb Technol, 1992, 14：321-326.
[13] O'Kennedy RD, Patching JW. Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and beta-galactosidase expression by Saccharomyces cerevisiae[J]. J Ind Microbiol Biotechnol, 1997, 18：319-325.
[14] O'Kennedy RD, Ward JM, Keshavarz-Moore E. Effects of fermentation strategy on the characteristics of plasmid DNA production[J]. Biotechnol Appl Biochem, 2003, 37：83-90.
[15] Adamcik J, Viglasky V, Valle F, et al. Effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability[J]. Electrophoresis, 2002, 23：3300-3309.
[16] Higgins CF, Dorman CJ, Stirling DA, et al. A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli[J]. Cell, 1988, 52：569-584.
[17] O'Kennedy RD, Baldwin C, Keshavarz-Moore E. Effects of growth medium selection on plasmid DNA production and initial processing steps[J]. J Biotechnol, 2000, 76：175-183.
[18] Freitas SS, Azzoni AR, Santos JA, et al. On the stability of plasmid DNA vectors during cell culture and purification[J]. Mol Biotechnol, 2007, 36：151-158.
[19] Pristas P, Ivan J, Javorsky P. Structural instability of small rolling circle replication plasmids from Selenomonas ruminantium[J]. Plasmid, 2010, 64：74-81.
[20] Oliveira PH, Prather KJ, Prazeres DM, Monteiro GA. Structural instability of plasmid biopharmaceuticals：challenges and implications[J]. Trends Biotechnol, 2009, 27：503-513.
[21] Sabido A, Martínez LM, de Anda R, et al. A novel plasmid vector designed for chromosomal gene integration and expression：Use for developing a genetically stable Escherichia coli melanin production strain [J]. Plasmid, 2012, 69：16-23.
[22] 苟斌全, 张嗣良, 储炬, 等. 增强组成型重组大肠杆菌质粒稳定性的发酵策略[J]. 化学与生物工程, 2008, 25(10)：23-26.
[23] Jeong SO, Shin SC, Jong-Kee Y, et al. Construction of various bacteriophage λ mutants for stable and efficient production of recombinant protein in Escherichia coli[J]. Process Biochemistry, 2007, 42 ：486-490.
[24] Vidal L, Pinsach J, Striedner G, et al. Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli[J]. J Biotechnol, 2008, 134：127-162.
[25] Mairhofer J, Pfaffenzeller I, Merz D, Grabherr R. A novel antibiotic free plasmid selection system：advances in safe and efficient DNA therapy[J]. Biotechnol J, 2008, 3：83-91.
[26] Goh S, Good L. Plasmid selection in Escherichia coli using an endogenous essential gene marker[J]. BMC Biotechnol, 2008, 8：61-69.
[27] Philip DS, Sarovich DS, Pemberton JM. pPSY：A vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli[J] . Plasmid, 2008, 60 ：53-58.
[28] Nikel PI, de Lorenzo V. Implantation of unmarked regulatory and metabolic modules in Gram-negative bacteria with specialised mini-transposon delivery vectors[J]. J Biotechnol, 2013, 163 ：143-154.
[29] 陆文渊, 成浩, 王丽鸳, 周健. 茶氨酸生物合成基因工程菌的质粒稳定性研究[J]. 茶叶科学, 2008, 28(2)：147-151.
[30] 王宏华, 凌红丽, 侯竹美, 等. 鸡γ-干扰素基因重组质粒在工程菌株中的稳定性研究[J]. 微生物学通报, 2008, 35(7)：1055-1058.
[31] 张刚, 杨光, 裴海生, 等. 质粒平均分配基因parDE甲酸脱氢酶NADH再生系统稳定性的影响[J]. 过程工程学报, 2008, 8(2)：345-349.
[32] Filomena S, Jo?o AQ, Fernanda CD. Evaluating metabolic stress and plasmid stability in plasmid DNA production by Escherichia coli[J]. Biotechnol Adv, 2012, 30 ：691-708.
[33] Sousa F, Prazeres DM, Queiroz JA. Improvement of transfection efficiency by using supercoiled plasmid DNA purified with arginine affinity chromatography[J]. J Gene Med, 2009, 11：79-88.
[34] Thumann G, Stocker M, Maltusch C, et al. High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor[J]. Gene Ther, 2010, 17：181-189.
[35] Yau SY, Keshavarz-Moore E, Ward J. Host strain influences on supercoiled plasmid DNA production in Escherichia coli：implications for efficient design of large-scale processes[J]. Biotechnol Bioeng, 2008, 101：529-572.
[36] Martí L, Enric S, Antoni C, et al. IPTG limitation avoids metabolic burden and acetic acid accumulation in induced fed-batch cultures of Escherichia coli M15 under glucose limiting conditions[J]. Biochem Eng J, 2013, 70：78-83.