Bacterial expression and functional characterization of a naturally occurring exon6-less preprochymosin cDNA

Chymosin (Rennin EC 3.4.23.4), an aspartyl proteinase, is the major proteolytic enzyme in the fourth stomach of the unweaned calf, and it is formed by proteolytic activation of its zymogene, prochymosin. Following the cloning of synthesized cDNAs on mRNA pools extracted from the mucosa of the calf fourth stomach, we have identified an alternatively spliced form of preprochymosin cDNA (AS6 preprochymosin). Sequencing data analysis showed that the exon six has been spliced out and, therefore the gene product is 114 bp shorter in length. In order to determine the biological significance of the AS6 preprochymosin, we expressed the encoding cDNA together with a complete chymosin cDNA in E. coli. Under the same expression conditions, we found at least a 5-fold higher expression of AS6 preprochymosin protein in comparison to a full-length recombinant chymosin. Protein prediction program analyses showed that the missing exon contain groups of amino acids with high hydrophobicity score. Therefore, the deletion of this exon may explain the higher expression of the recombinant product in E. coli. Most importantly, the biological activity of the purified AS6 preprochymosin, was confirmed in an assay of chymosin milk-clotting activity using the recombinant preprochymosin and commercial rennet as positive controls. The expression of the biologically active preprochymosin lacking exon 6 may have important implications on the existence of this splicing form of mRNA in vivo and on its biotechnological applications in cheese manufacture.