A novel and simple method for high-level production of reverse transcriptase from Moloney murine leukemia virus (MMLV-RT) in Escherichia coli

被引：10

作者：

Chen, Yu ^{[1
]}

Xu, Weiguo ^{[2
]}

Sun, Qiming ^{[2
]}

机构：

[1] Nanjing Agr Univ, Coll Hort, Nanjing 210095, Peoples R China

[2] ProbeGene Biotechnol Corp, Nanjing 210029, Peoples R China

来源：

BIOTECHNOLOGY LETTERS | 2009年 / 31卷 / 07期

关键词：

Chaperone; Enterokinase; Leukemia virus; Lower temperature; Reverse transcriptase; OVEREXPRESSION; EXPRESSION;

D O I：

10.1007/s10529-009-9977-5

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：

A novel protocol for producing recombinant Moloney murine leukemia virus (MMLV-RT) in Escherichia coli is reported. The optimized coding sequence for mature MMLV-RT was cloned into pET28a and over-expressed as an N-terminal His6-tagged fusion protein. An enterokinase (EK) recognition site was introduced between the His6-tag and MMLV-RT to release tag-free enzyme. Optimal expression of soluble His6-MMLV-RT was achieved by chaperone co-expression and lower temperature fermentation. The His6-tagged enzyme was first purified by Ni2+ affinity chromatography. The bound enzyme was then eluted by EK digestion and the eluate was purified on an anion-exchange Q column to remove DNA and EK. Twenty-one milligram MMLV-RT was obtained from 1 l of bacterial culture.

引用

页码：1051 / 1057

页数：7

共 10 条

[1] NASBA ISOTHERMAL ENZYMATIC INVITRO NUCLEIC-ACID AMPLIFICATION OPTIMIZED FOR THE DIAGNOSIS OF HIV-1 INFECTION [J].

KIEVITS, T ;

VANGEMEN, B ;

VANSTRIJP, D ;

SCHUKKINK, R ;

DIRCKS, M ;

ADRIAANSE, H ;

MALEK, L ;

SOOKNANAN, R ;

LENS, P .

JOURNAL OF VIROLOGICAL METHODS, 1991, 35 (03) :273-286

[2]

KIMMEL AR, 1987, METHOD ENZYMOL, V152, P307

[3] CLONING AND OVEREXPRESSION OF MOLONEY MURINE LEUKEMIA-VIRUS REVERSE-TRANSCRIPTASE IN ESCHERICHIA-COLI [J].

KOTEWICZ, ML ;

DALESSIO, JM ;

DRIFTMIER, KM ;

BLODGETT, KP ;

GERARD, GF .

GENE, 1985, 35 (03) :249-258

[4] Overexpression of trigger factor prevents aggregation of recombinant proteins in Escherichia coli [J].

Nishihara, K ;

Kanemori, M ;

Yanagi, H ;

Yura, T .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2000, 66 (03) :884-889

[5]

ROTH MJ, 1985, J BIOL CHEM, V260, P9326

[6]

SUN QM, 2005, PROTEIN EXPRES PURIF, V31, P311

[7] EXPRESSION OF ENZYMATICALLY ACTIVE REVERSE-TRANSCRIPTASE IN ESCHERICHIA-COLI [J].

TANESE, N ;

ROTH, M ;

GOFF, SP .

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1985, 82 (15) :4944-4948

[8]

Wang Xiansong, 2008, Sheng Wu Gong Cheng Xue Bao, V24, P903

[9] Comparison of the thermal stabilities of reverse transcriptases from avian myeloblastosis virus and Moloney murine leukaemia virus [J].

Yasukawa, Kiyoshi ;

Nemoto, Daisuke ;

Inouye, Kuniyo .

JOURNAL OF BIOCHEMISTRY, 2008, 143 (02) :261-268

[10] Expression of human cardiac-specific homeobox protein in Escherichia coli [J].

Zhao, JH ;

Xu, Z ;

Hua, ZC .

PROTEIN EXPRESSION AND PURIFICATION, 2000, 18 (03) :316-319

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