Expression of active human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli and characterisation of the recombinant enzyme.
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-alpha-D-ribosyl-1-pyrophosphate and guanine were compared with those of lymphoblast and erythrocyte HPRT.
Free
M L
ML
Department of Biochemistry, University of Queensland, Australia.
Gordon
R B
RB
Keough
D T
DT
Beacham
I R
IR
Emmerson
B T
BT
de Jersey
J
J
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005091:Exons; D015870:Gene Expression; D005796:Genes; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D004247:DNA; D008715:Methionine; D007041:Hypoxanthine Phosphoribosyltransferase