大肠杆菌cysE和cysM基因的克隆、原核表达及多克隆抗体的制备
|
摘要
山羊绒的主要组成成分是角蛋白,角蛋白同时又是多种氨基酸的组合体,其中含量最高的是谷氨酸和胱氨酸,胱氨酸是含硫氨基酸。羊等哺乳动物自身不能直接合成半胱氨酸,只能依靠反式硫化蛋氨酸间接的合成半胱氨酸。利用转基因技术,将半胱氨酸合成酶转移至绒山羊体内,使得羊体自身能够高效的合成半胱氨酸,对山羊绒品质的提高有极大促进作用。本研究旨在通过PCR扩增大肠杆菌(Escherichia coli)丝氨酸乙酰转移酶基因(cysE)和O-乙酰丝氨酸硫化氢解酶基因(cysM),构建质粒载体进行原核表达,并制备相应的蛋白多克隆抗体,为生产半胱氨酸合成酶转基因绒山羊奠定方法与技术基础。
(1)从大肠杆菌基因组DNA中扩增cysE基因,长度为822 bp,编码274个氨基酸残基,cysM基因全长912 bp,编码304个氨基酸残基,各为1个完整的开放阅读框。测序后得到的序列,与Genbank公布的cysE(8177668)和cysM(8176794)序列相比对,同源性达到100%。经基因重组成功构建原核表达载体pET32a-cysE和pET32a-cysM,并可在大肠杆菌BL21(DE3)中诱导后实现高效表达。
(2)原核表达载体pET32a-cysE和pET32a-cysM在大肠杆菌BL21(DE3)经1 mmol/L IPTG诱导,37℃下,表达6 h,目的蛋白表达量超过全部菌蛋白表达量的25%。通过Ni-Agrose亲和柱纯化,从细菌裂解上清液中纯化得到了cysE和cysM融合蛋白。SDS-PAGE结果显示,亲和柱纯化法得到的融合蛋白纯度可达90%,回收效率达60%。
(3)用纯化的cysE和cysM蛋白作为抗原免疫新西兰大耳白兔,制备多克隆抗体。通过酶联免疫吸附试验(ELISA)检测抗体效价,抗体滴度分别达到1:102.4K,表明纯化制备的多克隆抗体具有较强的免疫结合活性。同时,以兔血清为一抗,HRP标记的羊抗兔IgG为二抗,对得到的cysE和cysM融合蛋白进行Western blot检测,表明试验获得抗体对原核表达的cysE和cysM融合蛋白具有特异性反应。
通过本研究建立了大肠杆菌cysE和cysM基因的克隆、原核表达及多克隆抗体制备的方法与技术。为进一步研究半胱氨酸合成酶转基因绒山羊奠定了技术基础,具有重要理论与育种实践价值。
The major component of goat cashmere fiber is keratin, which is a variety of combination of amino acids. The highest contents of keratin are glutamic acid and cystine, and cystine is a sulfur-containing amino acids. Goat and other mammals can only synthesis cysteine indirectly from methionine viatrans-sulphuration. Using genetic engineering, the expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the goat. It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. This study focuses on cloning serine acetyltransferase gene (cysE) and O-acetylserine sulfhydrylase-B gene (cysM) which encoding cysteine synthetase in Escherichia coli, prokaryotic expressing the enzymes and preparing their antibodies, which as preliminary works for the production of transgenic cashmere goat.
(1) The cysE and cysM genes are amplified by PCR from Escherichia coli genomic DNA. The cysE gene is 822 bp, which encoding 274 amino acids, and cysM gene is 912 bp, which encoding 304 amino acids. Each one of them is a complete open reading frame. Sequences alignment between sequencing results and published Genbank cysE (8177668) and cysM (8176794) sequences shows 100% homology. Prokaryotic expression vectors pET32a-cysE and pET32a-cysM are successfully constructed and efficiently express in BL21 (DE3) after induced.
(2) Induce by 1 mmol/L IPTG, 37℃, 6h, prokaryotic expression vectors pET32a-cysE and pET32a-cysM efficiently express in Escherichia coli BL21 (DE3), the expression of target protein over all the bacterial protein by 25%. The cysE and cysM proteins are purified from Bacterial lysate by Ni-Agrose affinity purification. SDS-PAGE results show that over 60% of the proteins are recovered, and the purity of purified proteins is 90%.
(3) Using purified cysE and cysM proteins as antigens immunize New Zealand white rabbits to create polyclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) detection shows that antibodies titers reach 1:102 400, indicates polyclonal antibodies have a strong immune binding activity. At the same time, using rabbit serum as primary antibody, HRP labeled goat anti-rabbit IgG as secondary antibody, the Western blott result shows that the antibodies have specific responses to cysE and cysM proteins.
Cloning, prokaryotic expression and polyclonal antibody preparation methods and techniques of Escherichia coli cysE and cysM gene were successfully established. Identified and purified cysE and cysM protein from prokaryotic expression. The preparation of recombinant cysE, cysM and their polyclonal antibodies has provided reliable tools for the future study in the transgenic sheep of cysteine biosynthesis gene.
(1)从大肠杆菌基因组DNA中扩增cysE基因,长度为822 bp,编码274个氨基酸残基,cysM基因全长912 bp,编码304个氨基酸残基,各为1个完整的开放阅读框。测序后得到的序列,与Genbank公布的cysE(8177668)和cysM(8176794)序列相比对,同源性达到100%。经基因重组成功构建原核表达载体pET32a-cysE和pET32a-cysM,并可在大肠杆菌BL21(DE3)中诱导后实现高效表达。
(2)原核表达载体pET32a-cysE和pET32a-cysM在大肠杆菌BL21(DE3)经1 mmol/L IPTG诱导,37℃下,表达6 h,目的蛋白表达量超过全部菌蛋白表达量的25%。通过Ni-Agrose亲和柱纯化,从细菌裂解上清液中纯化得到了cysE和cysM融合蛋白。SDS-PAGE结果显示,亲和柱纯化法得到的融合蛋白纯度可达90%,回收效率达60%。
(3)用纯化的cysE和cysM蛋白作为抗原免疫新西兰大耳白兔,制备多克隆抗体。通过酶联免疫吸附试验(ELISA)检测抗体效价,抗体滴度分别达到1:102.4K,表明纯化制备的多克隆抗体具有较强的免疫结合活性。同时,以兔血清为一抗,HRP标记的羊抗兔IgG为二抗,对得到的cysE和cysM融合蛋白进行Western blot检测,表明试验获得抗体对原核表达的cysE和cysM融合蛋白具有特异性反应。
通过本研究建立了大肠杆菌cysE和cysM基因的克隆、原核表达及多克隆抗体制备的方法与技术。为进一步研究半胱氨酸合成酶转基因绒山羊奠定了技术基础,具有重要理论与育种实践价值。
The major component of goat cashmere fiber is keratin, which is a variety of combination of amino acids. The highest contents of keratin are glutamic acid and cystine, and cystine is a sulfur-containing amino acids. Goat and other mammals can only synthesis cysteine indirectly from methionine viatrans-sulphuration. Using genetic engineering, the expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the goat. It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. This study focuses on cloning serine acetyltransferase gene (cysE) and O-acetylserine sulfhydrylase-B gene (cysM) which encoding cysteine synthetase in Escherichia coli, prokaryotic expressing the enzymes and preparing their antibodies, which as preliminary works for the production of transgenic cashmere goat.
(1) The cysE and cysM genes are amplified by PCR from Escherichia coli genomic DNA. The cysE gene is 822 bp, which encoding 274 amino acids, and cysM gene is 912 bp, which encoding 304 amino acids. Each one of them is a complete open reading frame. Sequences alignment between sequencing results and published Genbank cysE (8177668) and cysM (8176794) sequences shows 100% homology. Prokaryotic expression vectors pET32a-cysE and pET32a-cysM are successfully constructed and efficiently express in BL21 (DE3) after induced.
(2) Induce by 1 mmol/L IPTG, 37℃, 6h, prokaryotic expression vectors pET32a-cysE and pET32a-cysM efficiently express in Escherichia coli BL21 (DE3), the expression of target protein over all the bacterial protein by 25%. The cysE and cysM proteins are purified from Bacterial lysate by Ni-Agrose affinity purification. SDS-PAGE results show that over 60% of the proteins are recovered, and the purity of purified proteins is 90%.
(3) Using purified cysE and cysM proteins as antigens immunize New Zealand white rabbits to create polyclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) detection shows that antibodies titers reach 1:102 400, indicates polyclonal antibodies have a strong immune binding activity. At the same time, using rabbit serum as primary antibody, HRP labeled goat anti-rabbit IgG as secondary antibody, the Western blott result shows that the antibodies have specific responses to cysE and cysM proteins.
Cloning, prokaryotic expression and polyclonal antibody preparation methods and techniques of Escherichia coli cysE and cysM gene were successfully established. Identified and purified cysE and cysM protein from prokaryotic expression. The preparation of recombinant cysE, cysM and their polyclonal antibodies has provided reliable tools for the future study in the transgenic sheep of cysteine biosynthesis gene.
引文
曹锡琳. 2006.山羊绒及其织物的功能整理.毛纺科技,1(1):25-27.
程善燕,张英杰. 2010.山羊绒生长机理及营养调控的研究进展.中国草食动物,(增刊1):63-65.
程琰,毛旭虎,王庆旭,余抒,刘艳青,张晓丽,宁亚蕾,邹全明. 2008.原核融合表达载体的设计、构建及应用.免疫学杂志,24(6):618-624.
达文致,达文政. 2009.山羊绒毛与绵羊奶化学成分研究.现代农业科技,5(5):208-209.
贾志海,张微,朱晓萍. 2009.山羊绒生长机理及生长调控技术.新农业,12:48-49.
蒋朋飞,曹金锁,柳超,赵海平,张德礼. 2010.猪新基因BCL-G_L的克隆与原核表达及其多克隆抗体的制备.细胞与分子免疫学杂志, 12:1238-1245.
刘博. 2007.大肠杆菌稳定期特异性启动子的筛选及pSP表达载体的构建. [硕士学位论文].大连:大连理工大学.
刘海斌,胡锐,蔡凤坤,史延平,戚磊,杨靖,卫功庆. 2010.蛋白水平对舍饲辽宁绒山羊产绒性能及消化代谢的影响.吉林农业大学学报,32(1):89-94.
宋振,王劼，安宁，杨振，雷鸣，郭泽坤. 2009.人SUMO-1基因原核表达、纯化及多克隆抗体制备与鉴定.免疫学杂志,25(2):125-129.
涂吉华,贾志海,那仁巴图,欧斌,张永斌. 2008.补饲不同蛋白质水平精料对放牧绒山羊生产性能的影响.草食家畜,2(2):33-37.
徐军,刘皙洁. 2010.日粮中添加半胱胺对辽宁绒山羊产绒性能的影响.中国畜牧兽医,37(7):210-212.
王宏博,高雅琴. 2007.影响绒山羊产绒性能和绒毛品质的因素及其研究进展.中国草食动物,27(4): 62-63.
魏云霞. 2008.绒山羊绒毛生长机理研究进展.中国草食动物, 28(3): 54-56.
甄玉国,卢德勋,马宁,王洪荣,刘世民. 2004.中国绒山羊蛋白质和氨基酸营养研究新进展.吉林农业大学学报,26(3):317-322.
Agren D, Schnell R, Oehlmann W, Singh M, Schneider G. 2008. Cysteine synthase (cysM) of mycobacterium tuberculosis is an o-phosphoserine sulfhydrylase evidence for an alternative cysteine biosynthesis pathway in mycobacteria. Journal of Biological Chemistry, 283(46): 31567-31574.
Bawden CS, Sivaprasad AV, Verma PJ, Walker SK, Rogers GE. 1995. Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep - toward a new in-vivo amino-acid biosynthesis pathway and improved wool growth. Transgenic Research, 4(2): 87-104.
Becker MA, Kredich NM, Tomkins GM. 1969. The purification and characterization of O-acetylserine sulfhydrylase-A from Salmonella typhimurium. Journal of Biological Chemistry, 244: 2418–2427.
Bettati S, Benci S, Campanini B, Raboni S, Chirico G, Beretta S, Schnackerz KD, Hazlett TL, Gratton E, Mozzarelli A. 2000. Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase. Journal of Biological Chemistry, 275: 40244-40251.
Bogdanova N, Hell R. 1997. Cysteine synthesis in plants: protein–protein interactions of serine acetyltransferase from Arabidopsis thaliana. The Plant Journal, 11: 251–262.
Borup B, Ferry JG. 2000. O-acetylserine sulfhydrylase from methanosarcina thermophila. Journal ofBacteriology, 182(1): 45-50.
Burkhard P, Rao GS, Hohenester E, Schnackerz KD, Cook PF, Jansonius JN. 1998. Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium. Journal of Molecular Biology, 283: 121–133.
Cao Z, Zhang J, Li Y, Xu X, Liu G, Bhattacharrya M K, Yang H, Ren D. 2007. Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits. Protein Expression and Purification, 52: 306-312.
Claus MT, Zocher GE, Maier TH, Schulz GE. 2005. Structure of the o-acetylserine sulfhydrylase isoenzyme cysM from Escherichia coli. Biochemistry, 44(24): 8620-8626.
Cook PF, Wedding RT. 1977. Initial kinetic characterization of the multienzyme complex, cysteine synthetase. Archives of Biochemistry and Biophysics, 178: 293–302.
Dubendorff JW, Studier FW. 1991. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology, 219(1): 45-59.
Duffaud GD, March PE, Inouye M. 1987. Expression and secretion of foreign proteins in Escherichia coli. Methods Enzymol, 153: 492-507.
Droux M, Martin J, Sajus P, Douce R. 1992. Purification and characterization of O-acetylserine(thiol)lyase from spinach chloroplasts. Archives of Biochemistry and Biophysics, 295: 379–390.
Feldman-Salit A, Wirtz M, Hell R, Wade RC. 2009. A mechanistic model of the cysteine synthase complex. Journal of Molecular Biology, 386(1): 37-59.
Galbraith H. 2000. Protein and sulphur amino acid nutrition of hair fibre-producing angora and cashmere goats. Livestock Production Science, 64(1): 81-93.
Garvis SG, Tipton SL, Konkel ME. 1997. Identification of a functional homolog of the Escherichia coli and Salmonella typhimurium cysM gene encoding O-acetylserine sulfhydrylase B in Campylobacter jejuni. Gene, 185 : 63–67.
Guo Y, Susan SW, Viswanath B. 2009. A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli. Protein Expression and Purification, 65: 230–237.
Hell R. 1997. Molecular physiology of plant sulfur metabolism. Planta, 202 (2) : 138–148.
Huang B, Vetting MW, Roderick SL. 2005. The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase. Journal of Bacteriology, 187: 3201–3205.
Ishikawa K, Mino K, Nakamura T. 2010. New function and application of the cysteine synthase from archaea. Biochemical Engineering Journal, 48(3): 315-322.
Knight KL, Tunyaplin C. 1995. Immunoglobulin heavy chain genes of rabbit. Immunoglobulin Genes: 289-314.
Kredich NM. 1996. Biosynthesis of cysteine, in: F.C. Neidhardt (Ed.), second edition., Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 1, American Society of Microbiology, Washington, DC., pp. 514–527.
Kredich NM, Tomkins GM. 1966. The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. Journal of Molecular Biology, 241: 4955–4965.
Kredich NM, Becker MA, Tomkins GM. 1969. Purification and characterization of cysteine synthetase, a bifunctional protein complex, from Salmonella typhimurium. Journal of Molecular Biology, 244 :2428–2439.
Kumar S, Raj I, Nagpal I, Subbarao N, Gourinath S. 2011. Structural and Biochemical Studies of Serine Acetyltransferase Reveal Why the Parasite Entamoeba histolytica Cannot Form a Cysteine Synthase Complex Journal of Molecular Biology, 286: 12533-12541.
Lin YW, Zhao DX, Wang ZH. 2006. Expression of lipase2 solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione transferase fusion protein(GST-cytb5). Protein Expression Purification, 45(2): 352-358.
Liu Q, Lin J, Liu M, Tao X, Wei D, Ma X, Yang S. 2007. Large scale preparation of recombinant human parathyroid hormone 1–84 from Escherichia coli. Protein Expression and Purification, 54: 212–219.
Ma H, Zhang W, Zhu XP, Song WH, Liu J, Jia ZH. 2010. Effects of rumen-protected tryptophan on growth performance, fibre characteristics, nutrient utilization and plasma essential amino acids in Cashmere goats during the cashmere slow growth period. Livestock Science, 131(2-3): 227-233.
Maier THP. 2003. Semisynthetic production of unnatural L--amino acids by metabolic engineering of the cysteine-biosynthetic pathway. Nature. Biotechnology, 21: 422–427.
McGregor BA. 2009. A review of cashmere nutrition experiments with suggestions for improving their design and conduct. Small Ruminant Research, 82(2-3): 71-83.
Michele G, Lorena P, Linda A, Rosita A, Massimo T, Cesare I. 2009. Over-expression in E.coli and purification of the human OCTN1 transport protein. Protein Expression and Purification, 68: 215–220.
Mierendorf RC, Morris BB, Hammer B, Novy RE. 1998. Expression and purification of recombinant proteins using the pET system.Methods in Molecular Medicine, 13: 257-292.
Mino K, Yamanoue T, Sakiyama T, Eisaki N, Mstsuyama A. 2000. Effects of bienzyme complex formation of cysteine synthetase from Escherichia coli on some properties and kinetics. Bioscience Biotechnology & Biochemistry, 64 : 1628–1640.
Mino K, Imamura K, Sakiyama T. 2001. Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex. Bioscience Biotechnology & Biochemistry, 65: 865–874.
Morgan JI, Curran T. 1995. Immediate-early genes: ten years on. Trends Neurosci, 18: 66–67.
Nakamura K, Tamura G. 1990. Isolation of serine acetyltransferase complexed with cysteine synthase from Allium tuberosum. Agricultural and biological chemistry, 54 : 649–656.
Nakamura T, Iwahashi H, Eguchi Y.1984. Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium. Journal of Bacteriology, 158: 1122–1127.
Roger AC, Carl H. 2009. An E.coli over-expression system for multiply-phosphorylated proteins and its use in a study of calcium phosphate sequestration by novel recombinant phosphopeptides. Protein Expression and Purification, 67: 23-34.
Robert M, Keith Y. 1994. The pET system: Your choice for expression. AdvancedProducts and Protoeols for Molecular Biology Research, 1(l): 3-36.
Saito K, Yokoyama H, Noji M, Murakoshi I. 1995. Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon. Journal of Molecular Biology, 270: 16321–16326.
Salsi E, Campanini B, Bettati S. 2010. A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex. Journal of Molecular Biology, 285: 12813-12822.
Salsi E, Guan R, Campanini B, Bettati S, Lin J, Cook PF, Mozzarelli A. 2011. Exploring o-acetylserine sulfhydrylase-b isoenzyme from salmonella typhimurium by fluorescence spectroscopy. Archives of Biochemistry and Biophysics, 505(2): 178-185.
Simionatto S, Marchioro SB, Galli V, Hartwig DD, Carlessi RM, Munari FM, Laurino JP, Conceicao FR, Dellagostin OA. 2010. Cloning and purification of recombinant proteins of Mycoplasma hyopneumoniae expressed in Escherichia coli. Protein Expression and Purification, 69: 132-136.
Simpanya MF, Leverenz VR, Giblin FJ. 2010. Expression and purification of his-tagged recombinant mouse-crystallin. Protein Expression and Purification, 69: 147-152.
Sirko A, Hryniewicz M, Hulanicka D. 1990. Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster. Journal of Bacteriology, 172: 3351–3357.
Sirko AE, Zatyka M, Hulanicka MD.1987. Identification of the Escherichia coli cysM gene encoding O-acetylserine sulphydrylase B by cloning with mini-Mu-lac containing a plasmid replicon. Journal of General Microbiology, 133: 2719–2725.
Tai CH, Nalabolu SR, Jacobson TM, Minter DE, Cook PF. 1993. Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternative reactants. Biochemistry, 32: 6433–6442.
Tai CH, Nalabolu SR, Simmons III JW. 1995. Acid–base chemical mechanism of O-acetylserine sulfhydrylases-A and -B from pH studies. Biochemistry, 34: 12311–12322.
Turnbull AL, Surette MG. 2010. Cysteine biosynthesis, oxidative stress and antibiotic resistance in salmonella typhimurium. Research in Microbiology, 161(8): 643-650.
Wirtz M, Droux M, Hell R. 2004. O-acetylserine(thiol)lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana. J Exp Bot, 55: 1785-1798.
Xiang D, Zhang J, Chen Y, Guo Y, Schalow A, Zhang Z, Hu X, Yu H, Zhao M, Zhu S, Lu H, Wu M, Yu Y, Moldenhauer A, Han W. 2010. Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli. Protein Expression and Purification, 69: 153-158.
Zhao C, Ohno H, Sogoh K, Imamura K, Sakiyama K, Nakanishi T. 2004. Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine. Journal of Bioscience and Bioengineering, 97(5): 322-328.
Zhao C, Kumada Y, Imanaka H, Imamuraa K, Nakanishi K. 2006. Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from escherichia coli. Protein Expression and Purification, 47(2): 607-613.
Zhou HM, Allain D, Li JQ, Zhang WG, Yu XC. 2003. Effects of non-genetic factors on production traits of Inner Mongolia cashmere goats in China. Small Ruminant Research, 47(1): 85-89.
程善燕,张英杰. 2010.山羊绒生长机理及营养调控的研究进展.中国草食动物,(增刊1):63-65.
程琰,毛旭虎,王庆旭,余抒,刘艳青,张晓丽,宁亚蕾,邹全明. 2008.原核融合表达载体的设计、构建及应用.免疫学杂志,24(6):618-624.
达文致,达文政. 2009.山羊绒毛与绵羊奶化学成分研究.现代农业科技,5(5):208-209.
贾志海,张微,朱晓萍. 2009.山羊绒生长机理及生长调控技术.新农业,12:48-49.
蒋朋飞,曹金锁,柳超,赵海平,张德礼. 2010.猪新基因BCL-G_L的克隆与原核表达及其多克隆抗体的制备.细胞与分子免疫学杂志, 12:1238-1245.
刘博. 2007.大肠杆菌稳定期特异性启动子的筛选及pSP表达载体的构建. [硕士学位论文].大连:大连理工大学.
刘海斌,胡锐,蔡凤坤,史延平,戚磊,杨靖,卫功庆. 2010.蛋白水平对舍饲辽宁绒山羊产绒性能及消化代谢的影响.吉林农业大学学报,32(1):89-94.
宋振,王劼，安宁，杨振，雷鸣，郭泽坤. 2009.人SUMO-1基因原核表达、纯化及多克隆抗体制备与鉴定.免疫学杂志,25(2):125-129.
涂吉华,贾志海,那仁巴图,欧斌,张永斌. 2008.补饲不同蛋白质水平精料对放牧绒山羊生产性能的影响.草食家畜,2(2):33-37.
徐军,刘皙洁. 2010.日粮中添加半胱胺对辽宁绒山羊产绒性能的影响.中国畜牧兽医,37(7):210-212.
王宏博,高雅琴. 2007.影响绒山羊产绒性能和绒毛品质的因素及其研究进展.中国草食动物,27(4): 62-63.
魏云霞. 2008.绒山羊绒毛生长机理研究进展.中国草食动物, 28(3): 54-56.
甄玉国,卢德勋,马宁,王洪荣,刘世民. 2004.中国绒山羊蛋白质和氨基酸营养研究新进展.吉林农业大学学报,26(3):317-322.
Agren D, Schnell R, Oehlmann W, Singh M, Schneider G. 2008. Cysteine synthase (cysM) of mycobacterium tuberculosis is an o-phosphoserine sulfhydrylase evidence for an alternative cysteine biosynthesis pathway in mycobacteria. Journal of Biological Chemistry, 283(46): 31567-31574.
Bawden CS, Sivaprasad AV, Verma PJ, Walker SK, Rogers GE. 1995. Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep - toward a new in-vivo amino-acid biosynthesis pathway and improved wool growth. Transgenic Research, 4(2): 87-104.
Becker MA, Kredich NM, Tomkins GM. 1969. The purification and characterization of O-acetylserine sulfhydrylase-A from Salmonella typhimurium. Journal of Biological Chemistry, 244: 2418–2427.
Bettati S, Benci S, Campanini B, Raboni S, Chirico G, Beretta S, Schnackerz KD, Hazlett TL, Gratton E, Mozzarelli A. 2000. Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase. Journal of Biological Chemistry, 275: 40244-40251.
Bogdanova N, Hell R. 1997. Cysteine synthesis in plants: protein–protein interactions of serine acetyltransferase from Arabidopsis thaliana. The Plant Journal, 11: 251–262.
Borup B, Ferry JG. 2000. O-acetylserine sulfhydrylase from methanosarcina thermophila. Journal ofBacteriology, 182(1): 45-50.
Burkhard P, Rao GS, Hohenester E, Schnackerz KD, Cook PF, Jansonius JN. 1998. Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium. Journal of Molecular Biology, 283: 121–133.
Cao Z, Zhang J, Li Y, Xu X, Liu G, Bhattacharrya M K, Yang H, Ren D. 2007. Preparation of polyclonal antibody specific for AtPLC4, an Arabidopsis phosphatidylinositol-specific phospholipase C in rabbits. Protein Expression and Purification, 52: 306-312.
Claus MT, Zocher GE, Maier TH, Schulz GE. 2005. Structure of the o-acetylserine sulfhydrylase isoenzyme cysM from Escherichia coli. Biochemistry, 44(24): 8620-8626.
Cook PF, Wedding RT. 1977. Initial kinetic characterization of the multienzyme complex, cysteine synthetase. Archives of Biochemistry and Biophysics, 178: 293–302.
Dubendorff JW, Studier FW. 1991. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology, 219(1): 45-59.
Duffaud GD, March PE, Inouye M. 1987. Expression and secretion of foreign proteins in Escherichia coli. Methods Enzymol, 153: 492-507.
Droux M, Martin J, Sajus P, Douce R. 1992. Purification and characterization of O-acetylserine(thiol)lyase from spinach chloroplasts. Archives of Biochemistry and Biophysics, 295: 379–390.
Feldman-Salit A, Wirtz M, Hell R, Wade RC. 2009. A mechanistic model of the cysteine synthase complex. Journal of Molecular Biology, 386(1): 37-59.
Galbraith H. 2000. Protein and sulphur amino acid nutrition of hair fibre-producing angora and cashmere goats. Livestock Production Science, 64(1): 81-93.
Garvis SG, Tipton SL, Konkel ME. 1997. Identification of a functional homolog of the Escherichia coli and Salmonella typhimurium cysM gene encoding O-acetylserine sulfhydrylase B in Campylobacter jejuni. Gene, 185 : 63–67.
Guo Y, Susan SW, Viswanath B. 2009. A novel bicistronic vector for overexpressing Mycobacterium tuberculosis proteins in Escherichia coli. Protein Expression and Purification, 65: 230–237.
Hell R. 1997. Molecular physiology of plant sulfur metabolism. Planta, 202 (2) : 138–148.
Huang B, Vetting MW, Roderick SL. 2005. The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase. Journal of Bacteriology, 187: 3201–3205.
Ishikawa K, Mino K, Nakamura T. 2010. New function and application of the cysteine synthase from archaea. Biochemical Engineering Journal, 48(3): 315-322.
Knight KL, Tunyaplin C. 1995. Immunoglobulin heavy chain genes of rabbit. Immunoglobulin Genes: 289-314.
Kredich NM. 1996. Biosynthesis of cysteine, in: F.C. Neidhardt (Ed.), second edition., Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, vol. 1, American Society of Microbiology, Washington, DC., pp. 514–527.
Kredich NM, Tomkins GM. 1966. The enzymic synthesis of L-cysteine in Escherichia coli and Salmonella typhimurium. Journal of Molecular Biology, 241: 4955–4965.
Kredich NM, Becker MA, Tomkins GM. 1969. Purification and characterization of cysteine synthetase, a bifunctional protein complex, from Salmonella typhimurium. Journal of Molecular Biology, 244 :2428–2439.
Kumar S, Raj I, Nagpal I, Subbarao N, Gourinath S. 2011. Structural and Biochemical Studies of Serine Acetyltransferase Reveal Why the Parasite Entamoeba histolytica Cannot Form a Cysteine Synthase Complex Journal of Molecular Biology, 286: 12533-12541.
Lin YW, Zhao DX, Wang ZH. 2006. Expression of lipase2 solubilized bovine liver microsomal cytochrome b5 in Escherichia coli as a glutathione transferase fusion protein(GST-cytb5). Protein Expression Purification, 45(2): 352-358.
Liu Q, Lin J, Liu M, Tao X, Wei D, Ma X, Yang S. 2007. Large scale preparation of recombinant human parathyroid hormone 1–84 from Escherichia coli. Protein Expression and Purification, 54: 212–219.
Ma H, Zhang W, Zhu XP, Song WH, Liu J, Jia ZH. 2010. Effects of rumen-protected tryptophan on growth performance, fibre characteristics, nutrient utilization and plasma essential amino acids in Cashmere goats during the cashmere slow growth period. Livestock Science, 131(2-3): 227-233.
Maier THP. 2003. Semisynthetic production of unnatural L--amino acids by metabolic engineering of the cysteine-biosynthetic pathway. Nature. Biotechnology, 21: 422–427.
McGregor BA. 2009. A review of cashmere nutrition experiments with suggestions for improving their design and conduct. Small Ruminant Research, 82(2-3): 71-83.
Michele G, Lorena P, Linda A, Rosita A, Massimo T, Cesare I. 2009. Over-expression in E.coli and purification of the human OCTN1 transport protein. Protein Expression and Purification, 68: 215–220.
Mierendorf RC, Morris BB, Hammer B, Novy RE. 1998. Expression and purification of recombinant proteins using the pET system.Methods in Molecular Medicine, 13: 257-292.
Mino K, Yamanoue T, Sakiyama T, Eisaki N, Mstsuyama A. 2000. Effects of bienzyme complex formation of cysteine synthetase from Escherichia coli on some properties and kinetics. Bioscience Biotechnology & Biochemistry, 64 : 1628–1640.
Mino K, Imamura K, Sakiyama T. 2001. Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex. Bioscience Biotechnology & Biochemistry, 65: 865–874.
Morgan JI, Curran T. 1995. Immediate-early genes: ten years on. Trends Neurosci, 18: 66–67.
Nakamura K, Tamura G. 1990. Isolation of serine acetyltransferase complexed with cysteine synthase from Allium tuberosum. Agricultural and biological chemistry, 54 : 649–656.
Nakamura T, Iwahashi H, Eguchi Y.1984. Enzymatic proof for the identity of the S-sulfocysteine synthase and cysteine synthase B of Salmonella typhimurium. Journal of Bacteriology, 158: 1122–1127.
Roger AC, Carl H. 2009. An E.coli over-expression system for multiply-phosphorylated proteins and its use in a study of calcium phosphate sequestration by novel recombinant phosphopeptides. Protein Expression and Purification, 67: 23-34.
Robert M, Keith Y. 1994. The pET system: Your choice for expression. AdvancedProducts and Protoeols for Molecular Biology Research, 1(l): 3-36.
Saito K, Yokoyama H, Noji M, Murakoshi I. 1995. Molecular cloning and characterization of a plant serine acetyltransferase playing a regulatory role in cysteine biosynthesis from watermelon. Journal of Molecular Biology, 270: 16321–16326.
Salsi E, Campanini B, Bettati S. 2010. A Two-step Process Controls the Formation of the Bienzyme Cysteine Synthase Complex. Journal of Molecular Biology, 285: 12813-12822.
Salsi E, Guan R, Campanini B, Bettati S, Lin J, Cook PF, Mozzarelli A. 2011. Exploring o-acetylserine sulfhydrylase-b isoenzyme from salmonella typhimurium by fluorescence spectroscopy. Archives of Biochemistry and Biophysics, 505(2): 178-185.
Simionatto S, Marchioro SB, Galli V, Hartwig DD, Carlessi RM, Munari FM, Laurino JP, Conceicao FR, Dellagostin OA. 2010. Cloning and purification of recombinant proteins of Mycoplasma hyopneumoniae expressed in Escherichia coli. Protein Expression and Purification, 69: 132-136.
Simpanya MF, Leverenz VR, Giblin FJ. 2010. Expression and purification of his-tagged recombinant mouse-crystallin. Protein Expression and Purification, 69: 147-152.
Sirko A, Hryniewicz M, Hulanicka D. 1990. Sulfate and thiosulfate transport in Escherichia coli K-12: nucleotide sequence and expression of the cysTWAM gene cluster. Journal of Bacteriology, 172: 3351–3357.
Sirko AE, Zatyka M, Hulanicka MD.1987. Identification of the Escherichia coli cysM gene encoding O-acetylserine sulphydrylase B by cloning with mini-Mu-lac containing a plasmid replicon. Journal of General Microbiology, 133: 2719–2725.
Tai CH, Nalabolu SR, Jacobson TM, Minter DE, Cook PF. 1993. Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternative reactants. Biochemistry, 32: 6433–6442.
Tai CH, Nalabolu SR, Simmons III JW. 1995. Acid–base chemical mechanism of O-acetylserine sulfhydrylases-A and -B from pH studies. Biochemistry, 34: 12311–12322.
Turnbull AL, Surette MG. 2010. Cysteine biosynthesis, oxidative stress and antibiotic resistance in salmonella typhimurium. Research in Microbiology, 161(8): 643-650.
Wirtz M, Droux M, Hell R. 2004. O-acetylserine(thiol)lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in Arabidopsis thaliana. J Exp Bot, 55: 1785-1798.
Xiang D, Zhang J, Chen Y, Guo Y, Schalow A, Zhang Z, Hu X, Yu H, Zhao M, Zhu S, Lu H, Wu M, Yu Y, Moldenhauer A, Han W. 2010. Expressions and purification of a mature form of recombinant human Chemerin in Escherichia coli. Protein Expression and Purification, 69: 153-158.
Zhao C, Ohno H, Sogoh K, Imamura K, Sakiyama K, Nakanishi T. 2004. Production of nonproteinaceous amino acids using recombinant Escherichia coli cells expressing cysteine synthase and related enzymes with or without the secretion of O-acetyl-L-serine. Journal of Bioscience and Bioengineering, 97(5): 322-328.
Zhao C, Kumada Y, Imanaka H, Imamuraa K, Nakanishi K. 2006. Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from escherichia coli. Protein Expression and Purification, 47(2): 607-613.
Zhou HM, Allain D, Li JQ, Zhang WG, Yu XC. 2003. Effects of non-genetic factors on production traits of Inner Mongolia cashmere goats in China. Small Ruminant Research, 47(1): 85-89.
© 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 |