Stuart R. Michelson

Affinity purification of the HIV-1 protease

An inhibitor of the HIV-1 protease has been employed in the generation of a resin which allows the rapid purification of this enzyme. A peptide substrate analogue, H2N-Ser-Gln-Asn-(Phe-psi[CH2N]-Pro)-Ile-Val-Gln-OH, was coupled to agarose resin. The HIV-1 protease was expressed in E. coli and the supernatant from lysed cells was passed through the affinity resin. Active HIV-1 protease was then eluted with a buffer change to pH 10 and 2 M NaCl. Final purification to a homogeneous preparation, capable of crystallization, was achieved with hydrophobic interaction chromatography. Solutions containing HIV-1 protease bound to competitive inhibitors do not bind to the column.

Polynucleotide phosphorylase covalently bound to cellulose and its use in the preparation of homopolynucleotides

Abstract A readily prepared, crude concentrate of polynucleotide phosphorylase (PNPase) has been covalently bound by the method of Porath to cyanogen bromide-activated cellulose; this is the first report of a synthesizing enzyme bound to a solid support. The bound enzyme had good activity and stability; more than forty consecutive polymerization cycles of nucleoside diphosphates have been carried out with a single preparation to give consistently good yields of stable polymers, and these were readily separated from the bound phosphorylase without tedious phenol extractions.