Complete secretion of activable bovine prochymosin by genetically engineered L forms of Proteus mirabilis

• Published in: Applied and Environmental Microbiology Vol. 55; no. 4; pp. 1009 - 1015
• Main Authors: Klessen, C, Schmidt, K.H, Gumpert, J, Grosse, H.H, Malke, H
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01-04-1989
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Animals; Biological and medical sciences; Biotechnology; Blotting, Western; Cattle; Chymosin - biosynthesis; Chymosin - genetics; Chymosin - metabolism; CHYMOSINE; Culture Media; DNA, Bacterial - genetics; Enzyme Precursors - biosynthesis; Enzyme Precursors - genetics; Enzyme Precursors - metabolism; FABRICACION DEL QUESO; Fermentation; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; gene transfer; Genetic engineering; Genetic technics; Genetic Vectors; L Forms - genetics; L Forms - metabolism; MEDIO DE CULTIVO; Methods. Procedures. Technologies; MILIEU DE CULTURE; Milk - metabolism; Plasmids; prochymosin; PROTEINAS; PROTEINE; PROTEUS; Proteus mirabilis; Proteus mirabilis - genetics; Proteus mirabilis - metabolism; QUIMOSINA; RECOMBINACION; RECOMBINAISON; Recombinant Proteins - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; SECRECION; SECRETION; SINTESIS DE PROTEINAS; SYNTHESE PROTEIQUE; TECHNOLOGIE FROMAGERE; Vectors (cloning, transfer, expression). Insertion sequences and transposons; Culture medium; Excretion; Bovine; Enzyme; Chymosin; Recombinant DNA; Gene expression; Conversion; Precursor; Vertebrata; Mammalia; Enzymatic activity; Immunoblotting assay; Bacteria; Genetic engineering; Proteus mirabilis; Artiodactyla; Ungulata; Nucleic acid; Enterobacteriaceae
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.55.4.1009-1015.1989

To circumvent problems encountered in the synthesis of active chymosin in a number of bacteria and fungi, a recombinant DNA L-form expression system that directed the complete secretion of fully activable prochymosin into the extracellular culture medium was developed. The expression plasmid constructions involved the in-frame fusion of prochymosin of cDNA minus codons 1 to 4 to streptococcal pyrogenic exotoxin type A gene (speA') sequences, including the speA promoter, ribosomal binding site, and signal sequence and five codons of mature SpeA. Secretion of fusion prochymosin enzymatically and immunologically indistinguishable from bovine prochymosin was achieved after transformation of two stable protoplast type L-form strains derived from Proteus mirabilis. The secreted proenzyme was converted by autocatalytic processing to chymosin showing milk-clotting activity. In controlled laboratory fermentation processes, a maximum specific rate of activable prochymosin synthesis of 0.57 X 10(-3)/h was determined from the time courses of biomass dry weight and product formation. Yields as high as 40 +/- 10 micrograms/ml were obtained in the cell-free culture fluid of strain L99 carrying a naturally altered expression plasmid of increased segregational stability. The expression-secretion system described may be generally useful for production of recombinant mammalian proteins synthesized intracellularly as aberrantly folded insoluble aggregates