Specific cleavage of hybrid proteins by proteinase encoded by the KEX2 gene.
A method for isolation of the KEX2-gene-encoded membrane-bound proteinase from alpha-cells of Saccharomyces cerevisiae yeast has been modified. The isolated enzyme hydrolyzes peptides and proteins with basic amino acid pairs which are cleaved at the C-ends of their peptide bonds. Because KEX2 proteinase is located within the Golgi compartment, it may be isolated by differential centrifugation of broken cells at 7000g for 15 min and at 20,000g for 15 min. By extracting the fraction that contains the active enzyme by a detergent solution, a protein has been obtained with specific activity 30 times higher than that of the membrane extract prepared according to the standard technique. This protocol decreases the number of steps required to isolate the enzyme. The effects of pH and inhibitors on KEX2 proteinase-catalyzed hydrolysis of Ac-Leu-Lys-Arg-pNA were studied. KEX2 proteinase can participate in peptide hormone processing because it cleaves human proinsulin at the peptide bond between Arg32 and Glu33. The KEX2 proteinase can specifically cleave large recombinant proteins, for example, a protein consisting of a gamma-interferon fragment linked to HIV1-proteinase via a Lys-Arg-containing peptide.
Bessmertnaya LYa
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. mila@enzyme.siobc.ras.ru
Loiko
I I
II
Goncharova
T I
TI
Ivanov
N V
NV
Rumsh
L D
LD
Antonov
V K
VK
D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013381:Subtilisins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae