Chemical synthesis of a biotinylated derivative of the simian immunodeficiency virus protease. Purification by avidin affinity chromatography and autocatalytic activation.
The protease from simian immunodeficiency virus (SIV) was chemically synthesized by automated solid-phase technology as an NH2-terminally extended derivative, capped with biotin. Biotin-linker-(SIV protease (1-99)): the linker segment, Gly-Gly-Asp-Arg-Gly-Phe-Ala-Ala, corresponds to the amino acid sequence preceding that of the protease in the SIV gag/pol precursor polyprotein. Accordingly, the Ala-Pro bond joining the octapeptide linker to the protease constitutes a site naturally cleaved by the protease during viral maturation. This strategy for synthesis was designed to facilitate purification of the biotinylated protein derivative from a complex mixture of reaction products by avidin/agarose-affinity chromatography and to provide the means for autocatalytic removal of the biotin-linker segment. As anticipated, folding of the full-length construct leads to activation of the enzyme and excision of the desired 99-residue SIV protease (overall yield, approximately). The specificity of the synthetic SIV protease toward a number of well characterized protein substrates was the same as observed for the nearly identical enzyme from human immunodeficiency virus type 2 (HIV-2 protease) and distinct from that of the more disparate HIV-1 protease. The same functional ordering with respect to the human retroviral proteases was reflected in Ki values observed with a number of protease inhibitors. Thus, the folded synthetic SIV protease shows patterns of specificity and susceptibility to inhibition that are in accord with what would be expected based upon its degree of structural similarity to proteases from HIV-1 and HIV-2.
Tomasselli
A G
AG
Biochemistry Unit, Upjohn Laboratories, Kalamazoo, Michigan 49001.
Bannow
C A
CA
Deibel
M R
MR
Hui
J O
JO
Zurcher-Neely
H A
HA
Reardon
I M
IM
Smith
C W
CW
Heinrikson
R L
RL
D000595:Amino Acid Sequence; D001360:Avidin; D001710:Biotin; D002384:Catalysis; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism; D017320:HIV Protease Inhibitors; D015497:HIV-1 / Q000201:enzymology; D015498:HIV-2 / Q000201:enzymology; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D015302:Simian Immunodeficiency Virus / Q000201:enzymology*; D013379:Substrate Specificity
D017320:HIV Protease Inhibitors; D011480:Protease Inhibitors; D001360:Avidin; D001710:Biotin; D010450:Endopeptidases; D016333:HIV Protease