黑曲霉β-葡萄糖苷酶基因在大肠杆菌中的克隆及表达研究
|
摘要
β-葡萄糖苷酶(EC3.2.1.21)属于糖苷水解酶家族3,它能够水解非还原性末端的β-D葡萄糖苷键,释放出游离的葡萄糖及相应的配基。β-葡萄糖苷酶是纤维素降解中的关键酶,对于可再生资源纤维素的利用具有十分重要的意义,但其在野生型宿主菌中的低含量及低酶活成为纤维素酶解的限制因素,因此通过基因工程手段实现外源高效表达β-葡萄糖苷酶已经成为各国学者研究的热点。黑曲霉是常见的安全菌株之一,同时也是普遍公认的含有β-葡萄糖苷酶较多的菌株,实现黑曲霉β-葡萄糖苷酶基因外源高效表达具有十分重要的意义。
本论文以总RNA为模板,通过逆转录PCR (RT-PCR)方法成功扩增了黑曲霉β-葡萄糖苷酶基因全长bgl序列,同时以基因组DNA为模板分别扩增出外显子5和6的序列E5,E6,PCR产物大小表明与预期一致。并成功构建三个重组表达质粒pET32a(+)-bgl, pET32a(+)-E5及pET28a(+)-E5。
将bgl及E5片段进行测序,结果表明E5大小为1192bp,bgl为2583bp,将DNA序列的E5片段与cDNA外显子5进行比对,发现其同源性达100%,表明所获得的bglcDNA序列是黑曲霉β-葡萄糖苷酶正确的开放阅读框,是由转录过程中DNA模板上的外显子拼接而成。6gl与5个来源于黑曲霉的β-葡萄糖苷酶基因同源性达99%,推测的氨基酸序列同源性达99%,蛋白质功能结构域预测结果显示其属于糖苷水解酶家族3。
将重组质粒转化大肠杆菌BL21(DE3)诱导表达,优化诱导条件之后,E5蛋白仍以包涵体形式存在于沉淀中。BGL可溶性表达略有增加,最佳表达条件为20℃,0.5mmol/L IPTG诱导12h,目的蛋白约占总蛋白的32.14%,可溶性部分约为15%,酶活29.8U／mg,约为野生型的30%。
β-glucosidase(EC3.2.1.21) belongs to glycoside hydrolases superfamily 3, it hydrolyses P-D glucosidic bond and releases glucose and corresponding aglucone.β-glucosidase is the key enzyme of cellulose degradation, and plays an important role in the utilization of renewable resources such as cellulose. However, the low concentration and low enzyme activity ofβ-glucosidase in wild-type strains are becoming the limiting factors during cellulose hydrolysis. Now more and more researchers are contributing to the cloning and expression ofβ-glucosidase in foreign expression systems with gene engineering strategy. Aspergillus niger is known as one of the common safe strains, as well as a strain producing relative highβ-glucosidase, so it is very significant to express Aspergillus nigerβ-glucosidase gene with high performance.
In this study, Aspergillus niger P-glucosidase gene bgl was amplified by reverse transcription PCR (RT-PCR) using the total RNA as template. Exon 5 and 6 sequences, E5 and E6 were amplified using the genome DNA as template.The PCR product of E5 and E6 were corresponded to the theoretical value. Three recombinant expression plasmids were then constructed:pET32a(+)-bgl, pET32a(+)-E5 and pET32a(+)-E5.
The sequencing results showed E5 was 1192bp, bgl was 2583bp, the sequence alignment showed that E5 sequence was 100% identical to the exon 5 region in cDNA sequence, which demonstrated the bgl gene was the right ORF forβ-glucosidase and it was formed by exons splicing. The bgl gene was 99% homologous to five Aspergillus niger P-glucosidase genes reported and the deduced amino acid sequence was 99%. Protein function region prediction showed BGL belonged to glycoside hydrolase superfamily 3.
Three recombinant expression plasmids were transformed into E.coli BL21(DE3) and induced by IPTG to expression. After optimization, E5 was still existed in the precipitation as inclusion bodies. BGL concentration was increased slightly in the supernatant. The optimized conditions for BGL expression were as follows:20℃,0.5mmol/L IPTG,12h. The whole target protein was 32.14% of the total and soluble protein was 15%.The recombinant enzyme activity was 29.8U/mg, which was 30% of the wild enzyme.
本论文以总RNA为模板,通过逆转录PCR (RT-PCR)方法成功扩增了黑曲霉β-葡萄糖苷酶基因全长bgl序列,同时以基因组DNA为模板分别扩增出外显子5和6的序列E5,E6,PCR产物大小表明与预期一致。并成功构建三个重组表达质粒pET32a(+)-bgl, pET32a(+)-E5及pET28a(+)-E5。
将bgl及E5片段进行测序,结果表明E5大小为1192bp,bgl为2583bp,将DNA序列的E5片段与cDNA外显子5进行比对,发现其同源性达100%,表明所获得的bglcDNA序列是黑曲霉β-葡萄糖苷酶正确的开放阅读框,是由转录过程中DNA模板上的外显子拼接而成。6gl与5个来源于黑曲霉的β-葡萄糖苷酶基因同源性达99%,推测的氨基酸序列同源性达99%,蛋白质功能结构域预测结果显示其属于糖苷水解酶家族3。
将重组质粒转化大肠杆菌BL21(DE3)诱导表达,优化诱导条件之后,E5蛋白仍以包涵体形式存在于沉淀中。BGL可溶性表达略有增加,最佳表达条件为20℃,0.5mmol/L IPTG诱导12h,目的蛋白约占总蛋白的32.14%,可溶性部分约为15%,酶活29.8U／mg,约为野生型的30%。
β-glucosidase(EC3.2.1.21) belongs to glycoside hydrolases superfamily 3, it hydrolyses P-D glucosidic bond and releases glucose and corresponding aglucone.β-glucosidase is the key enzyme of cellulose degradation, and plays an important role in the utilization of renewable resources such as cellulose. However, the low concentration and low enzyme activity ofβ-glucosidase in wild-type strains are becoming the limiting factors during cellulose hydrolysis. Now more and more researchers are contributing to the cloning and expression ofβ-glucosidase in foreign expression systems with gene engineering strategy. Aspergillus niger is known as one of the common safe strains, as well as a strain producing relative highβ-glucosidase, so it is very significant to express Aspergillus nigerβ-glucosidase gene with high performance.
In this study, Aspergillus niger P-glucosidase gene bgl was amplified by reverse transcription PCR (RT-PCR) using the total RNA as template. Exon 5 and 6 sequences, E5 and E6 were amplified using the genome DNA as template.The PCR product of E5 and E6 were corresponded to the theoretical value. Three recombinant expression plasmids were then constructed:pET32a(+)-bgl, pET32a(+)-E5 and pET32a(+)-E5.
The sequencing results showed E5 was 1192bp, bgl was 2583bp, the sequence alignment showed that E5 sequence was 100% identical to the exon 5 region in cDNA sequence, which demonstrated the bgl gene was the right ORF forβ-glucosidase and it was formed by exons splicing. The bgl gene was 99% homologous to five Aspergillus niger P-glucosidase genes reported and the deduced amino acid sequence was 99%. Protein function region prediction showed BGL belonged to glycoside hydrolase superfamily 3.
Three recombinant expression plasmids were transformed into E.coli BL21(DE3) and induced by IPTG to expression. After optimization, E5 was still existed in the precipitation as inclusion bodies. BGL concentration was increased slightly in the supernatant. The optimized conditions for BGL expression were as follows:20℃,0.5mmol/L IPTG,12h. The whole target protein was 32.14% of the total and soluble protein was 15%.The recombinant enzyme activity was 29.8U/mg, which was 30% of the wild enzyme.
引文
[1]武秀琴.纤维素酶及其应用.微生物学杂志.2009,29(2)：89-92
[2]牟晓红.生物质转化燃料乙醇纤维素酶的研究.石油化工应用.2009,28(3)：16-19
[3]张喜宏,刘义波等.纤维素酶及纤维素酶产生菌选育的研究进展.饲料工业.2009,30(22)：14-16
[4]王建荣,张曼夫等.绿色木酶纤维素酶CBHⅡ基因的结构研究.遗传学报.1995,22(1)：74-80
[5]张华锋.纤维素酶研究进展.现代农业科学.2009,16(6)：25-27
[6]顾卫民.β-葡萄糖苷酶的特性及其在食品工业中的应用.江苏食品与发酵.2003,18(1)：5-7
[7]李远华.β-葡萄糖苷酶的研究进展(综述).安徽农业大学学报.2002,29(4)：421-425
[8]孟宪文,宋小红等.β-葡萄糖苷酶的研究进展.乳品加工.2009,(10)：42-43
[9]Henrissat B. A classification of glycosyl-hydrolase based on amino acid sequence similarities. Biochem.1991(293):781-788
[10]王志江,魏江福等.β-葡萄糖苷酶的研究.饲料工业.2006,26(22)：20-22
[11]邓敏,邬小兵等.灰绿曲霉β-葡萄糖苷酶的分离及特性.生物技术.2009,19(6)：61-63
[12]杜娟,庄蕾等.棘孢曲霉SM-L22β-葡萄糖苷酶的纯化与性质.菌物系统.2002,21(2)：239-245
[13]孙迎庆,曹淑桂等.β-葡萄糖苷酶的分离纯化和性质研究.生物学杂志.1997,14(5)：12-15
[14]孟宪文,李长彪等.乳酸菌β-葡萄糖苷酶的分离纯化及特性研究.食品科学.2006,27(11)：116-119
[15]王冬梅,李多川等.嗜热子囊菌光孢变种Thermoascus aurantiacus var.levisporus β-葡萄糖苷酶的分离纯化及特性研究.农业环境科学学报.2005,24(5)：1007-1012
[16]S W Kengon, E J Luesink, et al. Purification and characterization of an extremely thermo stable beta-glucosidase from the hyperthermophilie archaeon Pyrococcus furiosus.European Journal of Biochemistry.1993,213:305-312
[17]潘利华,罗建平等.蚕豆β-葡萄糖苷酶的分离纯化及其性质研究.精深加工.2007(2)：88-91
[18]李志强,廖祥儒等.碧桃(Prunus persica L.)叶中β-葡萄糖苷酶的分离纯化和某些特性分析.植物生理学通讯.2007,43(2)：359-362
[19]J Pontoh, NH Low. Purification and characterization of Beta-glucosidase
from honeybees (Apis mellgera). Insect Biochemistry and Molecular Biology.2002,32: 679-690
[20]陈向东,藤尾雄策.日本根酶IF05318胞外β-葡萄糖苷酶的纯化及部分特性.微生物学报.1997,37(5)：368-373
[21]张喆,赵红等.福寿螺β-葡萄糖苷酶的分离纯化及性质的初步研究.厦门大学学报(自然科学版).1999,38(2)：287-291
[22]王沁,赵学慧.黑曲霉β-葡萄糖苷酶的纯化与性质.厦门大学学报(自然科学版).1992,32(6)：687-691
[23]彭喜春,彭志英.β-葡萄糖苷酶的研究现状及应用前景.江苏食品与发酵.2001(4)：22-25
[24]Moracci M, Nucci R, Febbraio F, et al. Expression and extensive characterization of a beta-glycosidase from the extreme thermoacidophilic archaeon sulfolobus solfataricus in escherichia coil:authenticity of the recombinant enzyme. Enzyme Microb Technol,1995, 17(11):992-997
[25]曾宇成,张树政.海枣曲霉β-葡萄糖苷酶的催化性质.微生物学报.1989,29(3)：195-199
[26]Jenkins J, Leggio L L, HarisG, et al. β-glucosidase, P-galactosidase, family A cellulases, family F xylanases and two barley glycanases from a superfamily of enzymes with a 8-fold a/parchitecture and with two conserved glutamates near the carboxyterminail ends of β-strands four and seven. FFBS Lett.1995,36(2):281-285
[27]Siegel Dan, Ira Marton. Cloning, expression, characterization, and Nucleophile Identification of family 3, Aspergillus niger β-glucosidase. The Journal of Biological Chemistry.2000,275(7):4973-4980
[28]Michae W. Bauer, Stephen G.W. Mechanism of Agrobacterium beta-glucosidase: kinetic studies.Biochemistry.1992,31(41):9961-9969
[29]Heather F. Seidle, Ira Marten. Physical and Kinetic Properties of the Family 3 β-Glucosidase from Aspergillus niger Which Is Important for Cellulose Breakdown. The Protein Journal.2004,23(1):11-23
[30]Walid Saibi, Bahia Amouri. Purification and biochemical characterization of a transglucosylating β-glucosidase of Stachybotrys strain. Appl Microbiol Biotechnol. 2007(77):293-300
[31]李华,高丽.β-葡萄糖苷酶活性测定方法的研究进展.食品与生物技术学报.2007,26(2)：107-114
[32]孙海燕.一种检测栀子蓝生成反应进程的新方法.化学与生物工程.2008,25(8)：64-66
[33]韩笑,陈介南等.β-葡萄糖苷酶基因的克隆与表达研究进展.生物技术通
报.2008(3)：8-12
[34]邹文等.黄单胞菌XA5-5 β-葡萄糖苷酶基因的克隆与表达.微生物学报.1994,34(4)：271-278
[35]赵云,刘伟丰等.多粘芽孢杆菌(Bacillus Polymyxa) β-葡萄糖苷酶基因在大肠杆菌中的表达、纯化及酶学性质分析.生物工程学报.2004,20(5)：741-744
[36]王让剑,江昌俊.两种茶树β-葡萄糖苷酶基因cDNA在原核中表达的研究.生物技术通报.2004(4)：102-105
[37]武汉大学,复旦大学生物系微生物学教研室编.微生物学.北京人民教育出版社.1990：448
[38]郭鲁宏,杨顺楷.黑曲霉产生的酶类及其应用.突然产物研究与开发.1997,10(4)：87-93
[39]Aniko Brumbauer. Study on heterogeneity of β-glucosidase from Aspergillus species by using counter-current distribution. Journal of Chromatography B.2000(743):247-254
[40]黑曲霉基因组测序计划完成.食品科学.2007,28(2)：176
[41]Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 1999(10):411-421
[42]Jonasson, P, Liljeqvist. S. Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol. Appl. Biochem.2002(35):91-105
[43]Mayer, M.P. A new set of useful cloning and expression vectors derived from pBlueScript. Gene.1995(163):41-46
[44]Connell, S.R., Tracz, D.M. Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrob.2003(47):3675-3681
[45]Hannig, G, Makrides, S.C. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol.1998(6):54-60
[46]Laursen, B.S., Steffensen, S. Structural requirements of the mRNA for intracistronic translation initiation of the enterobacterial infB gene. Genes Cells.2002(7):901-910
[47]Poole, E.S., Brown, CM. The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. EMBO J.1995(14): 151-158
[48]Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 1999(10):411-421
[49]Dubendorff, J.W., Studier, F.W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 1991(219):45-59
[50]Studier, F.W., Rosenberg, A.H. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol.1990(185):60-89
[51]Guzman, L.M., Belin, D. Tight regulation, modulation and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol.1995(177):4121-4130
[52]Calos, M.P. DNA sequence for a low-level promoter of the lac repressor gene and an 'up'promoter mutation. Nature.1978(274):762-765
[53]Studier, F.W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol.1991(219):37-44
[54]Khlebnikov, A., Keasling, J.D. Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers. Biotechnol. Prog. 2002(18):672-674
[55]pET System Manual. Novagen.2002
[56]D.H.Lee, H.S. Baek. Production of bone morphogenetic protein 7 using pET expression system. Current Applied Physics.2005:422-425
[57]Barbel Matthey. A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins. GENE.1999(229):145-153
[58]Camilla Giammarini. High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein. Protein Expression and Purification.2003(28):78-85
[59]Guzman, L.M., Belin, D. Tight regulation, modulation and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol.1995(177):4121^130
[60]Morgan-Kiss, R.M., Wadler, C. Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity. Proc. Natl. Acad. Sci. U. S. A.2002(99):7373-7377
[61]Siegele, D.A., Hu, J.C. Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proc. Natl. Acad. Sci. U. S. A.1997(94):8168-8172
[62]Englesberg, E., Squires, C. The 1-arabinose operon in Escherichia coli B-r:a genetic demonstration of two functional states of the product of a regulator gene. Proc. Natl. Acad. Sci. U. S. A.1969(62):1100-1107
[63]Chart, H., Smith, H.R. An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1. J. Appl. Microbiol.2000(89):1048-1058
[64]Yazdani S S, Mukherjee K J. Continuous culture studies on the stability and expression of recombinant streptokinase in Escherichia coli:Stability and expression of streptokinase in continuous culture. Bioprocess Biosyst Eng.2002,24(6):341-346
[65]Imanaka T, Aiba S. A perspective on the application of genetic engineering stability of recombinant plasmid. Ann N Y Acad Sci,1981,369(2):1-14
[2]牟晓红.生物质转化燃料乙醇纤维素酶的研究.石油化工应用.2009,28(3)：16-19
[3]张喜宏,刘义波等.纤维素酶及纤维素酶产生菌选育的研究进展.饲料工业.2009,30(22)：14-16
[4]王建荣,张曼夫等.绿色木酶纤维素酶CBHⅡ基因的结构研究.遗传学报.1995,22(1)：74-80
[5]张华锋.纤维素酶研究进展.现代农业科学.2009,16(6)：25-27
[6]顾卫民.β-葡萄糖苷酶的特性及其在食品工业中的应用.江苏食品与发酵.2003,18(1)：5-7
[7]李远华.β-葡萄糖苷酶的研究进展(综述).安徽农业大学学报.2002,29(4)：421-425
[8]孟宪文,宋小红等.β-葡萄糖苷酶的研究进展.乳品加工.2009,(10)：42-43
[9]Henrissat B. A classification of glycosyl-hydrolase based on amino acid sequence similarities. Biochem.1991(293):781-788
[10]王志江,魏江福等.β-葡萄糖苷酶的研究.饲料工业.2006,26(22)：20-22
[11]邓敏,邬小兵等.灰绿曲霉β-葡萄糖苷酶的分离及特性.生物技术.2009,19(6)：61-63
[12]杜娟,庄蕾等.棘孢曲霉SM-L22β-葡萄糖苷酶的纯化与性质.菌物系统.2002,21(2)：239-245
[13]孙迎庆,曹淑桂等.β-葡萄糖苷酶的分离纯化和性质研究.生物学杂志.1997,14(5)：12-15
[14]孟宪文,李长彪等.乳酸菌β-葡萄糖苷酶的分离纯化及特性研究.食品科学.2006,27(11)：116-119
[15]王冬梅,李多川等.嗜热子囊菌光孢变种Thermoascus aurantiacus var.levisporus β-葡萄糖苷酶的分离纯化及特性研究.农业环境科学学报.2005,24(5)：1007-1012
[16]S W Kengon, E J Luesink, et al. Purification and characterization of an extremely thermo stable beta-glucosidase from the hyperthermophilie archaeon Pyrococcus furiosus.European Journal of Biochemistry.1993,213:305-312
[17]潘利华,罗建平等.蚕豆β-葡萄糖苷酶的分离纯化及其性质研究.精深加工.2007(2)：88-91
[18]李志强,廖祥儒等.碧桃(Prunus persica L.)叶中β-葡萄糖苷酶的分离纯化和某些特性分析.植物生理学通讯.2007,43(2)：359-362
[19]J Pontoh, NH Low. Purification and characterization of Beta-glucosidase
from honeybees (Apis mellgera). Insect Biochemistry and Molecular Biology.2002,32: 679-690
[20]陈向东,藤尾雄策.日本根酶IF05318胞外β-葡萄糖苷酶的纯化及部分特性.微生物学报.1997,37(5)：368-373
[21]张喆,赵红等.福寿螺β-葡萄糖苷酶的分离纯化及性质的初步研究.厦门大学学报(自然科学版).1999,38(2)：287-291
[22]王沁,赵学慧.黑曲霉β-葡萄糖苷酶的纯化与性质.厦门大学学报(自然科学版).1992,32(6)：687-691
[23]彭喜春,彭志英.β-葡萄糖苷酶的研究现状及应用前景.江苏食品与发酵.2001(4)：22-25
[24]Moracci M, Nucci R, Febbraio F, et al. Expression and extensive characterization of a beta-glycosidase from the extreme thermoacidophilic archaeon sulfolobus solfataricus in escherichia coil:authenticity of the recombinant enzyme. Enzyme Microb Technol,1995, 17(11):992-997
[25]曾宇成,张树政.海枣曲霉β-葡萄糖苷酶的催化性质.微生物学报.1989,29(3)：195-199
[26]Jenkins J, Leggio L L, HarisG, et al. β-glucosidase, P-galactosidase, family A cellulases, family F xylanases and two barley glycanases from a superfamily of enzymes with a 8-fold a/parchitecture and with two conserved glutamates near the carboxyterminail ends of β-strands four and seven. FFBS Lett.1995,36(2):281-285
[27]Siegel Dan, Ira Marton. Cloning, expression, characterization, and Nucleophile Identification of family 3, Aspergillus niger β-glucosidase. The Journal of Biological Chemistry.2000,275(7):4973-4980
[28]Michae W. Bauer, Stephen G.W. Mechanism of Agrobacterium beta-glucosidase: kinetic studies.Biochemistry.1992,31(41):9961-9969
[29]Heather F. Seidle, Ira Marten. Physical and Kinetic Properties of the Family 3 β-Glucosidase from Aspergillus niger Which Is Important for Cellulose Breakdown. The Protein Journal.2004,23(1):11-23
[30]Walid Saibi, Bahia Amouri. Purification and biochemical characterization of a transglucosylating β-glucosidase of Stachybotrys strain. Appl Microbiol Biotechnol. 2007(77):293-300
[31]李华,高丽.β-葡萄糖苷酶活性测定方法的研究进展.食品与生物技术学报.2007,26(2)：107-114
[32]孙海燕.一种检测栀子蓝生成反应进程的新方法.化学与生物工程.2008,25(8)：64-66
[33]韩笑,陈介南等.β-葡萄糖苷酶基因的克隆与表达研究进展.生物技术通
报.2008(3)：8-12
[34]邹文等.黄单胞菌XA5-5 β-葡萄糖苷酶基因的克隆与表达.微生物学报.1994,34(4)：271-278
[35]赵云,刘伟丰等.多粘芽孢杆菌(Bacillus Polymyxa) β-葡萄糖苷酶基因在大肠杆菌中的表达、纯化及酶学性质分析.生物工程学报.2004,20(5)：741-744
[36]王让剑,江昌俊.两种茶树β-葡萄糖苷酶基因cDNA在原核中表达的研究.生物技术通报.2004(4)：102-105
[37]武汉大学,复旦大学生物系微生物学教研室编.微生物学.北京人民教育出版社.1990：448
[38]郭鲁宏,杨顺楷.黑曲霉产生的酶类及其应用.突然产物研究与开发.1997,10(4)：87-93
[39]Aniko Brumbauer. Study on heterogeneity of β-glucosidase from Aspergillus species by using counter-current distribution. Journal of Chromatography B.2000(743):247-254
[40]黑曲霉基因组测序计划完成.食品科学.2007,28(2)：176
[41]Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 1999(10):411-421
[42]Jonasson, P, Liljeqvist. S. Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol. Appl. Biochem.2002(35):91-105
[43]Mayer, M.P. A new set of useful cloning and expression vectors derived from pBlueScript. Gene.1995(163):41-46
[44]Connell, S.R., Tracz, D.M. Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrob.2003(47):3675-3681
[45]Hannig, G, Makrides, S.C. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol.1998(6):54-60
[46]Laursen, B.S., Steffensen, S. Structural requirements of the mRNA for intracistronic translation initiation of the enterobacterial infB gene. Genes Cells.2002(7):901-910
[47]Poole, E.S., Brown, CM. The identity of the base following the stop codon determines the efficiency of in vivo translational termination in Escherichia coli. EMBO J.1995(14): 151-158
[48]Baneyx, F. Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 1999(10):411-421
[49]Dubendorff, J.W., Studier, F.W. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 1991(219):45-59
[50]Studier, F.W., Rosenberg, A.H. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol.1990(185):60-89
[51]Guzman, L.M., Belin, D. Tight regulation, modulation and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol.1995(177):4121-4130
[52]Calos, M.P. DNA sequence for a low-level promoter of the lac repressor gene and an 'up'promoter mutation. Nature.1978(274):762-765
[53]Studier, F.W. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol.1991(219):37-44
[54]Khlebnikov, A., Keasling, J.D. Effect of lacY expression on homogeneity of induction from the P(tac) and P(trc) promoters by natural and synthetic inducers. Biotechnol. Prog. 2002(18):672-674
[55]pET System Manual. Novagen.2002
[56]D.H.Lee, H.S. Baek. Production of bone morphogenetic protein 7 using pET expression system. Current Applied Physics.2005:422-425
[57]Barbel Matthey. A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins. GENE.1999(229):145-153
[58]Camilla Giammarini. High-level expression of the Listeria monocytogenes listeriolysin O in Escherichia coli and preliminary characterization of the purified protein. Protein Expression and Purification.2003(28):78-85
[59]Guzman, L.M., Belin, D. Tight regulation, modulation and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol.1995(177):4121^130
[60]Morgan-Kiss, R.M., Wadler, C. Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity. Proc. Natl. Acad. Sci. U. S. A.2002(99):7373-7377
[61]Siegele, D.A., Hu, J.C. Gene expression from plasmids containing the araBAD promoter at subsaturating inducer concentrations represents mixed populations. Proc. Natl. Acad. Sci. U. S. A.1997(94):8168-8172
[62]Englesberg, E., Squires, C. The 1-arabinose operon in Escherichia coli B-r:a genetic demonstration of two functional states of the product of a regulator gene. Proc. Natl. Acad. Sci. U. S. A.1969(62):1100-1107
[63]Chart, H., Smith, H.R. An investigation into the pathogenic properties of Escherichia coli strains BLR, BL21, DH5alpha and EQ1. J. Appl. Microbiol.2000(89):1048-1058
[64]Yazdani S S, Mukherjee K J. Continuous culture studies on the stability and expression of recombinant streptokinase in Escherichia coli:Stability and expression of streptokinase in continuous culture. Bioprocess Biosyst Eng.2002,24(6):341-346
[65]Imanaka T, Aiba S. A perspective on the application of genetic engineering stability of recombinant plasmid. Ann N Y Acad Sci,1981,369(2):1-14
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |