EFFICIENT HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC SYSTEM FOR THE PURIFICATION OF A HALOBACTERIAL SERINE PROTEASE

Many of the extreme halophiles which belong to the archaebacteria produce extracellular proteases. The extracellular serine protease (designated ESP4) of Halobacterium sp. strain TuA4 was isolated in a pure state with a fast protein liquid chromatographic (FPLC) system. Because the enzyme is only stable at high ionic strength, it was necessary to develop a procedure that would allow a minimum Na+ ion concentration of 0.3 M in each step. This is the first halophilic salt-dependent enzyme purified with FPLC. Two precipitation steps with PEG 6000 and acetone in combination with ion-exchange chromatography (CM-Sephadex, Mono Q HR 5 5) and hydrophobic interaction chromatography (phenyl-Superose HR 5 5) permitted the isolation of 216-fold purified ESP4 with a total recovery of 3%. The purified ESP4 was shown to possess a molecular weight of 60 000 dalton in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, which correlates very well with the native molecular weight determined for this enzyme. © 1990.