Open AccessPurification of cysteine proteinases from trichomonads using bacitracin‐sepharose
Author(s) -
Irvine Joseph W.,
Coombs Graham H.,
North Michael J.
Publication year - 1993
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1993.tb06304.x
Subject(s) - tritrichomonas foetus , affinity chromatography , cysteine , biology , biochemistry , microbiology and biotechnology , peptide sequence , coomassie brilliant blue , sepharose , proteinase k , enzyme , staining , gene , fetus , pregnancy , genetics
Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100‐fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56‐fold more proteinase activity than was detectable in the original cell lysate was obtained from 10 9 cells (7.2 mg protein). SDS‐PAGE revealed that the eluate contained two main Coomassie blue‐staining bands. N‐terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N‐terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.