大肠杆菌无细胞体系合成异源蛋白质的初探
摘要
无细胞蛋白表达系统又称为体外翻译系统,是一种相对胞内表达系统而言的开放表达系统。它以外源mRNA或DNA为模板,在细胞抽提物的酶系中补充底物和能量来合成蛋白质。随着生物组学时代的到来,无细胞蛋白质合成系统显示出快速、方便、易于高通量等优点,因而应用范围不断增加。
大肠杆菌无细胞蛋白质合成系统是一种以外源mRNA或DNA为模板,利用大肠杆菌细胞抽提物的酶系,通过添加氨基酸、T7 RNA聚合酶和能量物质等来表达蛋白质的体外翻译系统。本论文开展大肠杆菌无细胞蛋白合成系统的构建工作,采用正交法对该系统进行优化,提高了大肠杆菌无细胞蛋白合成系统的合成目标蛋白质的能力。构建了6种HIV基因的大肠杆菌无细胞表达载体,并且选取HIV病毒感染因子(Vif)作为目标蛋白,在大肠杆菌无细胞蛋白质合成系统得到了表达,为将来高通量抗HIV药物得筛选奠定了一定的基础。最后,构建了适宜体外表达S-腺苷-L-甲硫氨酸合成酶和5-氨基乙酰丙酸合成酶的表达载体,为将来在无细胞体系中表达具有生物活性的酶开展了探索性工作。
The cell-free expression system is an in vitro translation system, compare with the in vivo translation system. Cell-free synthesis system enables the direct in vitro expression of protein from extrinsic template DNA or mRNA. It synthesizes protein through complementing substrate and energy in the cell extraction. In the new era of "bio-omics", the cell-free expression system opens a new strategy for protem expression, and is used widely because it can produce proteins in a mode of rapid, convenient and high-throughput expression.E. coli cell-free expression system is one widely used system which can synthesize protein effectively through complementing substrate and energy. The E. coli cell-free expression system was constructed and optimized in the present paper. The orthogal design method was used to improve the productivity in this E. coli cell-free system. Then six vectors were constructed for the purpose of expressing six HIV genes in E. coli cell-free system. Among them, VIF gene can be expressed effectively in this cell-free system. This work may be helpful to further expression of HIV genes in the cell-free system. Finally, two more genes (one SAM biosynthetic gene and one ALA biosynthetic gene) were inserted into cell-free vectors for the further expression of these two enzymes in the cell-free systems.
大肠杆菌无细胞蛋白质合成系统是一种以外源mRNA或DNA为模板,利用大肠杆菌细胞抽提物的酶系,通过添加氨基酸、T7 RNA聚合酶和能量物质等来表达蛋白质的体外翻译系统。本论文开展大肠杆菌无细胞蛋白合成系统的构建工作,采用正交法对该系统进行优化,提高了大肠杆菌无细胞蛋白合成系统的合成目标蛋白质的能力。构建了6种HIV基因的大肠杆菌无细胞表达载体,并且选取HIV病毒感染因子(Vif)作为目标蛋白,在大肠杆菌无细胞蛋白质合成系统得到了表达,为将来高通量抗HIV药物得筛选奠定了一定的基础。最后,构建了适宜体外表达S-腺苷-L-甲硫氨酸合成酶和5-氨基乙酰丙酸合成酶的表达载体,为将来在无细胞体系中表达具有生物活性的酶开展了探索性工作。
The cell-free expression system is an in vitro translation system, compare with the in vivo translation system. Cell-free synthesis system enables the direct in vitro expression of protein from extrinsic template DNA or mRNA. It synthesizes protein through complementing substrate and energy in the cell extraction. In the new era of "bio-omics", the cell-free expression system opens a new strategy for protem expression, and is used widely because it can produce proteins in a mode of rapid, convenient and high-throughput expression.E. coli cell-free expression system is one widely used system which can synthesize protein effectively through complementing substrate and energy. The E. coli cell-free expression system was constructed and optimized in the present paper. The orthogal design method was used to improve the productivity in this E. coli cell-free system. Then six vectors were constructed for the purpose of expressing six HIV genes in E. coli cell-free system. Among them, VIF gene can be expressed effectively in this cell-free system. This work may be helpful to further expression of HIV genes in the cell-free system. Finally, two more genes (one SAM biosynthetic gene and one ALA biosynthetic gene) were inserted into cell-free vectors for the further expression of these two enzymes in the cell-free systems.
引文
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4 J. Manley, G. Reiness, G. Zubay and Malcolm L. Gefter. Cell-free synthesis of SU~+m tyrosyl transfer RNA: Characterization of the 4S product. Archives of Biochemistry and Biophysics 1973;157: 50-54.
5 李前,孙曼霁.体外翻译系统在分子生物学研究中的应用.国外医学分子生物学分册,2002;24(1):18-19.
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9 Fessing. MY et al. terization of the cDNA encoding the murine, thiopurine S-methyltransferase(TPMT). FEBS Lett, 1998;424: 143.
10 Johnson VG, Nicholls PJ. Histidine 21 does not play a major role in diphtheria toxin catalysis. J Biol Chera, 1994;269: 4349.
11 金由辛,阮康成,包俊茹,龚佩娟,郑乐,王德宝.压力对兔网织红细胞蛋白质无细胞合成系统活力的影响.科学通报(Jin You-xin, Ruan Kang-cheng, Bao Jun-ru, Gong Pei-jiuan, Zheng le, Wang De-bao. The elect of pressure on activity of rabbit reticulocyte lysates. Chin Sci Bull), 1994;39(16): 1516-1518.
12 王润华,沈培奋,李钰云.一种简单快速微量的无细胞蛋白质合成系统.生物化学杂志 (Wang Run-hua, Shen Bei-fen, Li Yu-yun. A rapid and simplified microassay for measurement of protein synthesis in rabbit reticulocyte lysates. Chin Sci Bull), 1995;11(4): 430-433.
13 王景林,高墙基.微紫青CHBHI基因(Cbh I)在体外无细胞系统中的表达.中国生物化学和分子生物学报(Wang Jing-lin, Gao Pei-ji. In vitro expression of CBH I gene(Cbh I) of Penicilliura jamthinellum in coupled transcription-translation system. Chin J Biochem Mol Biol), 1999;15(6): 885-887.
14 徐志南,陈海琴,汪家权,岑沛霖.无细胞蛋白质合成系统的研究进展.中国生物化学与分子生物学报,2004;20(3):289-293.
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17 Busso D, Kim R, Kim SH. Expression of soluble recombinant proteins in a cell-free system using a 96-well format[J]. J Biochem Biophys Methods, 2003;55: 233-240.
18 Spirin A S, Baranow V I, Ryabova LA, Ovodov S Y, Alakhov Y B. A continuous cell-free translation system capable of producing polypeptides in high yield. Science, 1988;242: 1162-1164.
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21 Kim D M, Swartz J R. Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli Biotechnol Leu, 2000;22: 1537-1542.
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23 Kang, S. H., Oh, T. J., Kim, R. G, Kang, T. J., Hwang, S. H.,Lee, E. Y, Choi, C. Y., An efficient cell-free protein synthesis system using periplasmic phosphatase-removed S30 extract. J. Microbiol. Meth., 2000;43, 91-96.
24 Jermutus L, Ryabova LA, Pluckthun A. Recent advances in producing and selecting functional proteins by using cell-free translation. Curr Opin Biotech, 1998;9:534-548.
25 Hideki Tohda, Nobutoshi Chikazumi, Takuya Ueda, Kazuya Nishikawa and Kimitsuna Watanabe. Efficient expression of E. coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate mRNA. Journal of Biotechnology, 1994;34(1):61-69.
26 Arnold Brown and Stanley N. Cohen. Effects of X development on template specificity of Escherichia coli RNA polymerase. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974;335(2):123-138.
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31 Watzele M, Hofmann T, Nemetz C, Heindl D, Metzler T, Mutter W. Method for improving the stability of linear DNA in cell-free in vitro transcription/translation systems. US Patent 20030073201, 2003.
32 Kang T J, Woo J H, Song H K, Ahn J H, Kum J W, Han J, Choi C Y, Joo H. A cell-free protein synthesis system as an investigational tool for the translation stop processes. FEBS Leu, 2002;517:211-214.
33 He M, Taussing M J. DiscernArray? technology:a cell-free method for the generation of
protein arrays from PCR DNA.. J Immunol Methods, 2003;274:265-270.
34 Katanaev V L, Spirin A S, Reuss M, Siemann M. Formation of bacteriophage MS2 infectious units in a cel-free translation system. FEBS Leu, 1996;397:143-148
35 Chekulayeva M N, Kumasov O V, Shirokov V A, Spirin A S. Continuous-exchange cell-free protein-synthesizing system:Synthesis of HIV-1 antigen Nef. Biochem Biophys Res Commun, 2001;280:914-917.
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2 Collins. Cell-free synthesis of proteins coding for mobilisation functions of ColE1 and transposition functions of Tn3. J Gene, 1979;6: 29.
3 Noren CJ, et al. A general method for site-specific incorporation of unnatural amino acids into proteins. Science, 1989;244: 182.
4 J. Manley, G. Reiness, G. Zubay and Malcolm L. Gefter. Cell-free synthesis of SU~+m tyrosyl transfer RNA: Characterization of the 4S product. Archives of Biochemistry and Biophysics 1973;157: 50-54.
5 李前,孙曼霁.体外翻译系统在分子生物学研究中的应用.国外医学分子生物学分册,2002;24(1):18-19.
6 Pelham H, Jackson R. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem, 1976;67: 247.
7 Doyle K. Protocols and appLications guide(3rd ed). Promega Corporation, Printed in USA. 1992.
8 Daigle I, Li C. apl-1, a Caenorhabditis elegans Gene Encoding a Protein Related to the Human β-Amyloid Protein Precursor. Pore Natl Acad Sci USA, 1993;90: 12045.
9 Fessing. MY et al. terization of the cDNA encoding the murine, thiopurine S-methyltransferase(TPMT). FEBS Lett, 1998;424: 143.
10 Johnson VG, Nicholls PJ. Histidine 21 does not play a major role in diphtheria toxin catalysis. J Biol Chera, 1994;269: 4349.
11 金由辛,阮康成,包俊茹,龚佩娟,郑乐,王德宝.压力对兔网织红细胞蛋白质无细胞合成系统活力的影响.科学通报(Jin You-xin, Ruan Kang-cheng, Bao Jun-ru, Gong Pei-jiuan, Zheng le, Wang De-bao. The elect of pressure on activity of rabbit reticulocyte lysates. Chin Sci Bull), 1994;39(16): 1516-1518.
12 王润华,沈培奋,李钰云.一种简单快速微量的无细胞蛋白质合成系统.生物化学杂志 (Wang Run-hua, Shen Bei-fen, Li Yu-yun. A rapid and simplified microassay for measurement of protein synthesis in rabbit reticulocyte lysates. Chin Sci Bull), 1995;11(4): 430-433.
13 王景林,高墙基.微紫青CHBHI基因(Cbh I)在体外无细胞系统中的表达.中国生物化学和分子生物学报(Wang Jing-lin, Gao Pei-ji. In vitro expression of CBH I gene(Cbh I) of Penicilliura jamthinellum in coupled transcription-translation system. Chin J Biochem Mol Biol), 1999;15(6): 885-887.
14 徐志南,陈海琴,汪家权,岑沛霖.无细胞蛋白质合成系统的研究进展.中国生物化学与分子生物学报,2004;20(3):289-293.
15 Pratt JM. Coupled transcription-translation in prokaryotic cell-free systems.[A]. in: Hames, BD, Higgins SJ. Transcription and translation: a practical approach.[C] IRL Press, New York, 1984;179-209.
16 Makeyev EV, Kolb VA, Spirin AS. Cell-free immunology:construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire[J]. FEBS Letters, 1999;444: 177-180.
17 Busso D, Kim R, Kim SH. Expression of soluble recombinant proteins in a cell-free system using a 96-well format[J]. J Biochem Biophys Methods, 2003;55: 233-240.
18 Spirin A S, Baranow V I, Ryabova LA, Ovodov S Y, Alakhov Y B. A continuous cell-free translation system capable of producing polypeptides in high yield. Science, 1988;242: 1162-1164.
19 Kim D M, Swartz J R. Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotechnol Bioeng, 2001;74(4): 309-316.
20 Michael C, Jewett, James R. Swartz. Mimicking the Escherichia coil Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis. BIOTECHNOLOGYAND BIOENGINEERING, 2004;86(1): 19-26.
21 Kim D M, Swartz J R. Oxalate improves protein synthesis by enhancing ATP supply in a cell-free system derived from Escherichia coli Biotechnol Leu, 2000;22: 1537-1542.
22 Kim R G, Choi C Y. Expression-independent consumption of substrates in cell-free expression system from Escherichia coil. J Biotech, 2000;84: 27-32.
23 Kang, S. H., Oh, T. J., Kim, R. G, Kang, T. J., Hwang, S. H.,Lee, E. Y, Choi, C. Y., An efficient cell-free protein synthesis system using periplasmic phosphatase-removed S30 extract. J. Microbiol. Meth., 2000;43, 91-96.
24 Jermutus L, Ryabova LA, Pluckthun A. Recent advances in producing and selecting functional proteins by using cell-free translation. Curr Opin Biotech, 1998;9:534-548.
25 Hideki Tohda, Nobutoshi Chikazumi, Takuya Ueda, Kazuya Nishikawa and Kimitsuna Watanabe. Efficient expression of E. coli dihydrofolate reductase gene by an in vitro translation system using phosphorothioate mRNA. Journal of Biotechnology, 1994;34(1):61-69.
26 Arnold Brown and Stanley N. Cohen. Effects of X development on template specificity of Escherichia coli RNA polymerase. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, 1974;335(2):123-138.
27 Kobatake E, Ebisawa A, Asaka 0, Yanagida Y, Ikariyama Y, Aizawa M. Stabilization and translation of immobilized mRNA on latex beads for cell-free protein synthesis system. Appl Biochem Biotech, 1999;76:217-227.
28 Nishimura N, Kitaoka Y, Niwano M. Utilization of copper ion as a ribonuclease inhibitor in a cell-free protein synthesis system. J Ferment Bioeng, 1994;78:130-133.
29 Nakano H, Tanaka T, Kawarasaki Y, Yamane T. Highly productive cell-free protein synthesis system using condensed wheat-germ extract. J Biotechnol, 1996;46:275-282.
30 Jung G Y, Lee E Y, Kim Y E, jung B W, Kang S H, Choi C Y. Stabilization effect of zeolite on DHFR mRNA in a wheat germ cell-free protein synthesis system. Biosci Bioeng, 2000;89(2): 193-195.
31 Watzele M, Hofmann T, Nemetz C, Heindl D, Metzler T, Mutter W. Method for improving the stability of linear DNA in cell-free in vitro transcription/translation systems. US Patent 20030073201, 2003.
32 Kang T J, Woo J H, Song H K, Ahn J H, Kum J W, Han J, Choi C Y, Joo H. A cell-free protein synthesis system as an investigational tool for the translation stop processes. FEBS Leu, 2002;517:211-214.
33 He M, Taussing M J. DiscernArray? technology:a cell-free method for the generation of
protein arrays from PCR DNA.. J Immunol Methods, 2003;274:265-270.
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