Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage.
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.
Evans
D B
DB
Biochemistry and Cancer and Infectious Diseases Research, Upjohn Company, Kalamazoo, Michigan 49001.
Tarpley
W G
WG
Sharma
S K
SK
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006678:HIV / Q000201:enzymology / Q000235:genetics; D054303:HIV Reverse Transcriptase; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012083:Renin / Q000737:chemistry; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D001616:beta-Galactosidase; D012083:Renin