Isolation, biochemical characterization and crystallization of the p15gag proteinase of myeloblastosis associated virus expressed in E. coli
| Autor: | Vladimír Kostka, F. Kaprálek, Iva Pichová, Juraj Sedlacek, Vladimir Benes, S. Foundling, Petr Štrop, Milan Souček, L. Pavlíčková, M. Trávníček |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Cytoplasm
Sequence analysis Molecular Sequence Data Gene Products gag Biology Biochemistry Inclusion bodies law.invention law Escherichia coli Aspartic Acid Endopeptidases Amino Acid Sequence Isoelectric Point Peptide sequence Chromatography High Pressure Liquid chemistry.chemical_classification Avian Myeloblastosis Virus Rous sarcoma virus Base Sequence Fast protein liquid chromatography biology.organism_classification Molecular biology Recombinant Proteins Enzyme Isoelectric point chemistry DNA Viral Recombinant DNA Electrophoresis Polyacrylamide Gel Crystallization Protein Processing Post-Translational |
| Zdroj: | International Journal of Biochemistry. 24:235-242 |
| ISSN: | 0020-711X |
| DOI: | 10.1016/0020-711x(92)90252-v |
| Popis: | 1. 1. The p15 gag proteinase responsible for the processing of the polyprotein precursor of the myeloblastosis associated virus was obtained by a recombinant technique in an E. coli expression system. The massive expression of the intentionally truncated precursor (pr25 lac-Δgag ) was accompanied by its structurally correct processing. 2. 2. Three procedures for the purification of the recombinant proteinase from both the cytoplasmic fraction and the inclusion bodies were developed. 3. 3. The purified proteinase was compared with the authentic proteinase isolated from MAV virions by N -terminal sequence analysis and amino acid analysis, molecular weight determination, reverse-phase HPLC and FPLC elution profiles, electrophoretic mobility and isoelectric point determination, and activity assays with proteins and synthetic substrates. The identity of both enzymes was shown. 4. 3. Contrary to reported data, the amino acid sequence of the p15 gag proteinase diners from the sequence of the homologous Rous sarcoma virus proteinase in one residue only, as follows from cDNA sequencing. 5. 4. Crystallization of the proteinase from a citrate-phosphate buffer at pH 5.6 afforded hexagonal crystals which diffracted well at 2.3 A without deterioration. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|