Bacterial expression and purification of C1C2 domain of human factor VIII

ABSTRACT

The cDNA from the mycoparasitic fungus Trichoderma atroviride PTCC5220 encoding a 42 kDa chitinase [Chit42] was isolated. The nucleotide sequence of the cDNA fragment as having a 1263 bp open reading frame that encodes a 421 amino acid polypeptide, and a high homology was found with other reported Chit42 belonging to the Trichoderma sp. The 22 amino acid N-terminal sequence is a putative signal pep-tide for the possible secretion of the protein. The protein has been expressed and secreted as a mature form in Escherichia coll BL21[DE3] using the pelB leader sequence. The E. coll strain expressed Chit42 in an active form and secreted the protein into the medium. This recombinant chitinase has been shown to have inhibitory activity on mycelial growth and also, lytic activity on the cell wall of Rhizoctonia solani [AG2-2], causal agent of root rot in sugar beet in vitro. Expressed chitinase was optimally active at pH 5 and at 40°C. It is thermally stable at 60°C for more than 120 min at pH5