Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis
| Autor: | Craig A. Rosen, Chein-Hwa Chen, Reiner L. Gentz |
|---|---|
| Rok vydání: | 1989 |
| Předmět: |
Genes
Viral medicine.medical_treatment Molecular Sequence Data Biology Transfection Virus Replication Cell Line law.invention law Gene expression Escherichia coli medicine Amino Acid Sequence RNA Messenger Peptide sequence Histidine Messenger RNA Multidisciplinary Protease HIV Biological activity Oncogene Proteins Viral Molecular biology Genes Biochemistry Gene Products tat Recombinant DNA tat Gene Products Human Immunodeficiency Virus Transcription Factors Research Article |
| Zdroj: | Proceedings of the National Academy of Sciences. 86:821-824 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.86.3.821 |
| Popis: | Expression of the human immunodeficiency virus tat-encoded protein (Tat) is required for virus replication. A genetic approach was used to facilitate the purification of biologically active Tat. A recombinant Tat protein containing a stretch of six histidine residues and a protease cleavage site was engineered and purified to greater than 95% homogeneity in a single step by immobilized metal-ion chromatography with a special affinity resin that has selectivity for proteins with neighboring histidine residues. A modified scrape loading method for introduction of protein into cell monolayers was used to demonstrate that the purified Tat retained biological activity. Tat function was completely blocked in the presence of transcription inhibitors, which demonstrates the requirement of ongoing mRNA synthesis for trans-activation. These studies indicate that the mechanism of trans-activation is unlikely to involve a direct action of Tat on mRNA stability, transport, or translation and provides the basis for a rapid assay that can be used to identify inhibitors of trans-activation. The methods described herein should be useful for the functional analysis of other proteins that do not confer activity through a receptor-mediated pathway. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|