HIGH-LEVEL EXPRESSION OF ESCHERICHIA-COLI TRANSFER-RNA (M5U54)-METHYLTRANSFERASE

被引：13

作者：

GU, XR ^{[1
]}

SANTI, DV ^{[1
]}

机构：

[1] UNIV CALIF SAN FRANCISCO, DEPT PHARMACEUT CHEM, SAN FRANCISCO, CA 94143 USA

来源：

DNA AND CELL BIOLOGY | 1990年 / 9卷 / 04期

关键词：

D O I：

10.1089/dna.1990.9.273

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

A cloning and high-expression system for tRNA (m5U54)-methyltransferase (RUMT) is described. Polymerase chain reaction (PCR) was used to replicate the coding sequence and create flanking restriction sites for cloning. The PCR product was then inserted into expression vectors containing the tac and PL promoters. With the PL promoter, induced cells produced about 1.5% of their soluble protein as catalytically active RUMT. With the tac promoter, up to 8% of the total cell protein was active enzyme, and RUMT was purified to near homogeneity in three steps. © 1990, Mary Ann Liebert, Inc. All rights reserved.

引用

页码：273 / 278

页数：6

共 14 条

[1]

BJORK GR, 1975, J BACTERIOL, V124, P99

[2] ROLE OF RIBOTHYMIDINE IN THE THERMAL-STABILITY OF TRANSFER-RNA AS MONITORED BY PROTON MAGNETIC-RESONANCE [J].

DAVANLOO, P ;

SPRINZL, M ;

WATANABE, K ;

ALBANI, M ;

KERSTEN, H .

NUCLEIC ACIDS RESEARCH, 1979, 6 (04) :1571-1581

[3] MOLECULAR-CLONING OF THE PLASMID RP4 PRIMASE REGION IN A MULTI-HOST-RANGE TACP EXPRESSION VECTOR [J].

FURSTE, JP ;

PANSEGRAU, W ;

FRANK, R ;

BLOCKER, H ;

SCHOLZ, P ;

BAGDASARIAN, M ;

LANKA, E .

GENE, 1986, 48 (01) :119-131

[4]

GREENBERG R, 1980, J BIOL CHEM, V255, P8296

[5] ON THE ROLE OF RIBOSYLTHYMINE IN PROKARYOTIC TRANSFER-RNA FUNCTION [J].

KERSTEN, H ;

ALBANI, M ;

MANNLEIN, E ;

PRAISLER, R ;

WURMBACH, P ;

NIERHAUS, KH .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1981, 114 (02) :451-456

[6] SPECTROPHOTOMETRIC AND TURBIDIMETRIC METHODS FOR MEASURING PROTEINS [J].

LAYNE, E .

METHODS IN ENZYMOLOGY, 1957, 3 :447-454

[7]

Maniatis T., 1982, MOL CLONING

[8] GROWTH RATE-DEPENDENT REGULATION OF TRANSFER RIBONUCLEIC-ACID (5-METHYLURIDINE)METHYLTRANSFERASE IN ESCHERICHIA-COLI B-R [J].

NY, T ;

BJORK, GR .

JOURNAL OF BACTERIOLOGY, 1980, 141 (01) :67-73

[9] PURIFICATION OF TRANSFER-RNA (M5U54)-METHYLTRANSFERASE FROM ESCHERICHIA-COLI ASSOCIATION WITH RNA [J].

NY, T ;

LINDSTROM, PHR ;

HAGERVALL, TG ;

BJORK, GR .

EUROPEAN JOURNAL OF BIOCHEMISTRY, 1988, 177 (03) :467-475

[10] PRIMER-DIRECTED ENZYMATIC AMPLIFICATION OF DNA WITH A THERMOSTABLE DNA-POLYMERASE [J].

SAIKI, RK ;

GELFAND, DH ;

STOFFEL, S ;

SCHARF, SJ ;

HIGUCHI, R ;

HORN, GT ;

MULLIS, KB ;

ERLICH, HA .

SCIENCE, 1988, 239 (4839) :487-491

← 1 2 →