Synthesis of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli.
Naturally occurring mutations in hypoxanthine-guanine phosphoribosyltransferase (HPRT) have been identified by amino acid sequencing, cDNA cloning, and direct nucleotide sequencing of PCR-amplified transcripts. To determine the effect these mutations have on the catalytic properties of the molecule, knowledge of the three-dimensional structure of HPRT is required. A prerequisite for this, however, is the availability of a large amount of purified product for crystallization and x-ray diffraction analysis. For these reasons we have developed an effective means of producing high levels of human HPRT in Escherichia coli using the expression cassette PCR. By taking advantage of a T7 polymerase/promoter system, we have expressed both normal and variant human hprt sequences in E. coli. The proteins synthesized from these sequences are immunologically and enzymatically active, and are physically indistinguishable from the HPRT in B-lymphoblasts derived from normal and three HPRT-deficient subjects.
Davidson
B L
BL
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109.
Brown
J E
JE
Weber
C H
CH
Palella
T D
TD
Roessler
B J
BJ
D001483:Base Sequence; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013997:Time Factors
D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase