Studies on bovine chymotrypsin gene

Abstract

The program of protein similarity searches, FASTP, was run on an IBM PC microcomputer to search proteins evolutionarily related to chymotrypsion. The compared chymotrypsion sequence was a bovine chymotrypsinogen A and the protein sequences searched in program were the 10,527 sequences in the National Biomedical Research Foundation library. Most of the proteins which were shown to be evolutionarily related to bovine chymotrypsinogen A were members of the serine protease family. As in the case of bovine carboxypeptidase A, however, some highly similar proteins to bovine chymotrypsinogen A were not serine proteases. To get more informations on the evolution of chymotrypsion-related proteins, 7 proteins from the chymotrpsin-related proteins were multi-aligned by using a computer program, VAlign, which was run on a Cray 2S supercomputer. The result showed that there is an ancestral protein common to chymotrypsion-related proteins. The molecular cloning of cDNA encoding bovine chymotrypsion was performed to study chymotrypsion at the level of gene. The bovine $\alpha$ -chymotrypsion purified by CM-cellulose column chromatography was used as immunogen to produce anti-chymotrypsion antibodies in rabbits. The anti-chymotrypsion antibodies were further purified by passing through Sepharose 4B column conjugated with bovine $\alpha$ chymotrpsin. The affinity purified antibodies were used as probe in screening bovine pancreas cDNA library constructed in lambda gtll. In the first screening, several positively signaling plaques were isolated. But further identification of isolated plaques revealed that the signals of these plaques were not of true.