| Expression and characterization of the reverse transcriptase enzyme from type 1 human immunodeficiency virus using different baculoviral vector systems. | |
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MedLine Citation:
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PMID: 8575439 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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To produce the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in amounts required to study its structure and function, the p66 enzyme subunit was expressed using two different baculovirus vectors in Sf158 insect host cells. Both vectors permitted an efficient HIV-1 RT expression. The resulting products were purified up to 90% homogeneity, characterized, and investigated for their susceptibility to digestion with various proteases. The recombinant baculoviral RT obtained with the pAc373 expression vector was purified as a p66/p60 heterodimer. The recombinant His-RT was expressed with the pBlueBacHis vector. Thereby, the protein was tagged with an N-terminal hexahistidine peptide and it was purified as a p70/p70 homodimer. The two enzymes differed in their specific activity, kinetic properties, and in vitro activation by viral and non-viral proteases. The recombinant His-RT exhibited a lower specific activity than the recombinant RT. The latter yielded enzyme activities as high as an Escherichia coli-expressed RT. Removal of the hexahistidine tag from the recombinant His-RT by digestion with enterokinase resulted in a complete loss of enzyme activity. Thus, the hexahistidine tag might be an intrinsic part of the active recombinant His-RT. |
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Authors:
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K Pekrun; H Petry; K D Jentsch; D Moosmayer; G Hunsmann; W Lüke |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: European journal of biochemistry / FEBS Volume: 234 ISSN: 0014-2956 ISO Abbreviation: Eur. J. Biochem. Publication Date: 1995 Dec |
Date Detail:
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Created Date: 1996-03-11 Completed Date: 1996-03-11 Revised Date: 2007-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0107600 Medline TA: Eur J Biochem Country: GERMANY |
Other Details:
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Languages: eng Pagination: 811-8 Citation Subset: IM; X |
Affiliation:
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Department for Virology and Immunology, German Primate Centre, Göttingen, Germany. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Baculoviridae / genetics* Blotting, Western Cells, Cultured Chymotrypsin / metabolism DNA Primers Gene Expression Genetic Vectors* HIV Protease / metabolism HIV Reverse Transcriptase HIV-1 / enzymology* Humans Insects Kinetics Protein Folding Protein Processing, Post-Translational RNA-Directed DNA Polymerase / chemistry, genetics*, isolation & purification, metabolism* Recombinant Proteins / chemistry, isolation & purification, metabolism Trypsin / metabolism |
| Chemical | |
Reg. No./Substance:
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0/DNA Primers; 0/Recombinant Proteins; EC 2.7.7.49/HIV Reverse Transcriptase; EC 2.7.7.49/RNA-Directed DNA Polymerase; EC 3.4.21.1/Chymotrypsin; EC 3.4.21.4/Trypsin; EC 3.4.23.-/HIV Protease |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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