Synthesis of normal and variant human hypoxanthine-guanine phosphoribosyltransferase in Escherichia coli

• Published in: Gene Vol. 123; no. 2; pp. 271 - 275
• Main Authors: Davidson, Beverly L., Brown, Jennifer E., Weber, Christian H., Palella, Thomas D., Roessler, Blake J.
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 01.01.1993; Amsterdam Elsevier; New York, NY
• Subjects: B-lymphoblast; Base Sequence; Biological and medical sciences; crystallization; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene expression; Genetic Vectors; HPRT; Humans; Hypoxanthine Phosphoribosyltransferase - biosynthesis; Hypoxanthine Phosphoribosyltransferase - isolation & purification; Molecular and cellular biology; Molecular genetics; Molecular Sequence Data; Mutation; PCR; Polymerase Chain Reaction - methods; recombinant DNA; Recombinant Proteins - biosynthesis; Recombinant Proteins - isolation & purification; Time Factors; x-ray diffraction analysis; x-ray diffraction analysis; aa; SDS; DTT; denotes plasmidcarrier state; nt; PRPP; ECPCR; PAGE; HPRT; bp; SD; B-lymphoblast; kb; crystallization; IPTG; recombinant DNA; wt; PCR; Human; Heterologous system; Enzyme; Gene product; Escherichia coli; Hypoxanthine phosphoribosyltransferase; Gene expression; Polymerase chain reaction; Enzymatic activity; Bacteria; Mutation; Enterobacteriaceae; Polymorphism
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(93)90137-R

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.