Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase
| Autor: | W. Gary Tarpley, Nancy A. Strakalaitis, Debasish Chattopadhyay, David P. Brunner, H.M. Einspahr, Martin R. Deibel |
|---|---|
| Rok vydání: | 1992 |
| Předmět: |
Recombinant Fusion Proteins
Size-exclusion chromatography Molecular Sequence Data medicine.disease_cause law.invention Sepharose law medicine Escherichia coli Amino Acid Sequence RNase H Polymerase chemistry.chemical_classification Chromatography biology Chemistry RNA-Directed DNA Polymerase Chromatography Ion Exchange Reverse transcriptase HIV Reverse Transcriptase Enzyme Biochemistry biology.protein Recombinant DNA Chromatography Gel HIV-1 Electrophoresis Polyacrylamide Gel Biotechnology |
| Zdroj: | Protein expression and purification. 3(2) |
| ISSN: | 1046-5928 |
| Popis: | HIV-1 reverse transcriptase (RT) has been successfully expressed as a biologically active recombinant protein in Escherichia coli and purified to homogeneity. After partial purification, RT was obtained primarily in a heterodimeric form represented by two subunits of 66 and 51 kDa, but the preparation also included several forms distinguishable in size and charge by chromatography on ionic-exchange and gel-filtration columns. We have developed a purification method that yields a single heterodimeric form of RT. Our strategy involves the selection of RT molecules exhibiting uniformity in elution from QAE Sepharose anion-exchange columns and Superose 12 gel-filtration columns. In the former, RT is resolved into multiple peaks on the basis of enzymatic activity, one of which represents highly active and pure p66:p51 heterodimeric RT. This highly active RT fraction, after gel-filtration chromatography, yields a compositionally pure protein product free of observable microheterogeneity by 1D and 2D polyacrylamide gel electrophoresis under a variety of conditions. Furthermore, the RNAse H enzymatic activity associated with HIV-1 RT has been demonstrated to coelute with the purified polymerase activity during gel filtration at a size (120 kDa) consistent with its location on the heterodimeric protein molecule. |
| Databáze: | OpenAIRE |
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