Abstract:
:A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro-Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography.
journal_name
Protein Expr Purifjournal_title
Protein expression and purificationauthors
Evans DB,Tarpley WG,Sharma SKdoi
10.1016/1046-5928(91)90073-rsubject
Has Abstractpub_date
1991-04-01 00:00:00pages
205-13issue
2-3eissn
1046-5928issn
1096-0279pii
1046-5928(91)90073-Rjournal_volume
2pub_type
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