EXPRESSION OF ACTIVE HUMAN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE IN ESCHERICHIA-COLI AND CHARACTERIZATION OF THE RECOMBINANT ENZYME

被引：17

作者：

FREE, ML

GORDON, RB

KEOUGH, DT

BEACHAM, IR

EMMERSON, BT

DEJERSEY, J

机构：

[1] UNIV QUEENSLAND,DEPT BIOCHEM,BRISBANE,QLD 4072,AUSTRALIA

[2] UNIV QUEENSLAND,PRINCESS ALEXANDRA HOSP,DEPT MED,WOOLLOONGABBA,QLD,AUSTRALIA

[3] UNIV QUEENSLAND,CTR MOLEC BIOL & BIOTECHNOL,ST LUCIA,QLD 4067,AUSTRALIA

[4] GRIFFITH UNIV,DIV SCI & TECHNOL,NATHAN,QLD 4111,AUSTRALIA

来源：

BIOCHIMICA ET BIOPHYSICA ACTA | 1990年 / 1087卷 / 02期

基金：

澳大利亚研究理事会; 英国医学研究理事会;

关键词：

Gene expression; Gout; Human erythrocyte; Hypoxanthine-guanine phosphoribosyltransferase; Lesch-Nyhan; Recombinant enzyme;

D O I：

10.1016/0167-4781(90)90206-H

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 plasmid, pRG1, has been constructed by incorporating the coding sequence of human hypoxanthine-guanine phosphoribosyltransferase (HPRT) into the expression vector pT7-7. Expression of human HPRT has been achieved in HPRT- Escherichia coli cells transformed with pRG1 and pGP1-2, as shown by: (1) exclusive labelling with [35S]methionine of a polypeptide with the same mobility as purified human HPRT on SDS-PAGE; and (2) measurement of HPRT activity after cell lysis. Although the majority of the recombinant HPRT was present in the particulate fraction after cell lysis and centrifugation, sufficient HPRT activity was present in the supernatant fraction to allow comparison with the HPRT purified from human erythrocytes and the activity in human haemolysates and lymphoblast lysates. Small differences in electrophoretic mobility on native gels were found between HPRT activity from these sources. The Km values of recombinant HPRT for the substrates 5-phospho-α-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT. © 1990.

引用

页码：205 / 211

页数：7

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