Expression of an autoprocessing cat-HIV-1 proteinase fusion protein: Purification to homogeneity of the released 99 residue proteinase
| Autor: | Douglas S. Montgomery, Norman M. Gray, Adrian N. Hobden, Onkar M. P. Singh, Colin W. Dykes, Malcolm Peter Weir |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Chloramphenicol O-Acetyltransferase
Isopropyl Thiogalactoside Sequence analysis Recombinant Fusion Proteins Molecular Sequence Data Restriction Mapping Biophysics Gene Expression lac operon Biology medicine.disease_cause Biochemistry Androgen-Binding Protein Residue (chemistry) HIV Protease Escherichia coli medicine Amino Acid Sequence Enzyme kinetics Cloning Molecular Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification Base Sequence Cell Biology Chromatography Ion Exchange biology.organism_classification Molecular biology Enterobacteriaceae Fusion protein Recombinant Proteins Molecular Weight Kinetics Enzyme chemistry Chromatography Gel HIV-1 Electrophoresis Polyacrylamide Gel Oligonucleotide Probes Plasmids |
| Zdroj: | Biochemical and Biophysical Research Communications. 175:784-794 |
| ISSN: | 0006-291X |
| Popis: | The 99 residue human immunodeficiency virus type 1 proteinase has been expressed in Escherichia coli as part of an autocleaving fusion protein. Expression of the fusion protein is toxic to the host cells, however yields of the released proteinase have been improved by optimising induction and harvest times to increase culture biomass, and decrease degradation of the proteinase. Soluble proteinase was extracted from these cells by a simple and highly efficient three step process. N-terminal sequence analysis confirms that the enzyme preparation is highly pure and correctly autoprocessed. The proteinase cleaves peptide substrate IGCTLNFPISPIETV between F and P at pH 6.0 with a Km of 310μM and a Kcat of 14s −1 . The enzyme is sensitive to its ionic environment, showing stimulation of activity at high salt concentrations, and shows a pH optimising 5.5. |
| Databáze: | OpenAIRE |
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