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Amplified expression of streptomyces endo-beta-N-acetylglucosaminidase H in Escherichia coli and characterization of the enzyme product.

1985; Elsevier BV; Volume: 260; Issue: 9 Linguagem: Inglês

10.1016/s0021-9258(18)89077-9

1083-351X

Robert Trumbly, Phillips W. Robbins, Marlene Belfort, F. D. Ziegler, Frank Maley, Robert B. Trimble,

Microbial Metabolites in Food Biotechnology

was used to transform an E. coli strain lysogenic for a X prophage containing a temperature-sensitive repressor.By shifting cultures of pKCE3 lysogens to 42 "C, the production of Endo H commenced and was linear for about 1 h.Enzyme yields were amplified 150-fold above those obtained from comparable cultures of S. plicatus and represented 3 to 4% of total cellular protein, which enabled purification of Endo H to homogeneity by a rapid fourstep procedure.Although most of the cloned Endo H was secreted into the periplasmic space by E. coli, its 4 kDa leader sequence peptide (Robbins et al. (1984)) was only partially removed during processing.As a result the purified pKCE3 Endo H was a heterogeneous population of molecules with an average molecular mass of 31 kDa compared to the 28.9 kDa fully processed product normally secreted by S. plicatus.Despite the residual -2 kDa of leader sequence on the cloned pKCE3 product, there were no detectable differences in either the substrate specificity or the stability characteristics of the enzyme purified from E. coli or from S. plicatus.Of particular value for studies on glycoproteins was the finding that the genetically engineered Endo H was completely free of proteolytic contaminants.