Prochymosin expression in Bacillus subtilis.
Prochymosin (PC) sequence was cloned in Bacillus subtilis using two kinds of plasmid constructions. In plasmid pSM316 the cDNA was inserted to obtain the intracellular expression of the enzyme. The enzyme turned out to be expressed in an insoluble form which could be converted to native enzyme under proper denaturing and refolding conditions. The levels of intracellular expression of PC were further enhanced by modifying the 5' region of the gene in a way that a two-cistron expression system was created. For the PC secretion, the cDNA was fused to the subtilisin leader sequence and expressed under the control of the B. subtilis neutral protease promoter. A properly folded PC was secreted by the cells, although to low levels.
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Genetic Engineering and Microbiology Laboratory, Eniricerche S.p.A., Milan, Italy.
de Ferra
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Galli
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Grandi
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D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D012085:Chymosin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D004792:Enzyme Precursors; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; C032443:prorennin; D012085:Chymosin