title: Autoprocessing of the human immunodeficiency virus type 1 protease precursor expressed in Escherichia coli from a synthetic gene.
abstract: A gene encoding an N-terminally extended precursor of 107 residues of the human immunodeficiency virus type 1 protease (PR107) was chemically synthesized and cloned into a bacterial expression vector, under the control of the araB promoter. PR107 was expressed alone or fused in phase to the amino or carboxy terminus of the bacterial beta-galactosidase (beta-gal). The yield of protease and beta-gal was found to be significantly higher when the gene for PR107 was cloned upstream of the Escherichia coli lacZ gene (PR107-beta-gal). Comparisons of the level of cloned protein expression between protease precursor and mature form suggested that this enhanced expression was due to the additional 5' sequence of the PR107 gene, and occurred at the post-transcriptional level. Autoprocessing of protease precursor and its release from the beta-gal fusion protein were analysed using wild-type and mutated cleavage sites. Mutations were introduced at amino acids downstream of the F-P scissile bond, at positions P4' and P5' in the C-terminal site (TLNF*PISP), and at position P3' in a consensus N-terminal site (TLNF*PQITL) placed at the protease-beta-gal junction. The data obtained suggested that (i) autoprocessing at the carboxy-terminal F-P bond was not significantly influenced by the presence of the N-terminal precursor sequence, (ii) P4' and P5' substitutions in the C-terminal site had no effect on cleavage, and (iii) P3' in the N-terminal site tolerated a wide variety of substitutions.
authors.affiliation: Centre de Transfert en Biotechnologie-Microbiologie, CNRS-UA544, INSA, Toulouse, France.
authors.forename: V
authors.initials: V
authors.lastname: Valverde
authors.forename: P
authors.initials: P
authors.lastname: Lemay
authors.forename: J M
authors.initials: JM
authors.lastname: Masson
authors.forename: B
authors.initials: B
authors.lastname: Gay
authors.forename: P
authors.initials: P
authors.lastname: Boulanger
mesh_terms: D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005813:Genes, Synthetic / Q000235:genetics*; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D007763:Lac Operon; D008565:Membrane Proteins; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D012697:Serine Endopeptidases; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D016333:HIV Protease