SYNTHESIS AND SECRETION OF RECOMBINANT PENICILLIN-G ACYLASE IN BACTERIAL L-FORMS

L-form strains of Proteus mirabilis and Escherichia coli lacking the c ell wall represent an alternative prokaryotic cell system for the prod uction of recombinant proteins (KLESSEN et al. 1988, LAPLACE et al. 19 88a, 1989b). We could demonstrate that they are also able to synthesiz e the enzyme penicillin G acylase (PAC)(1)). PAC was processed and sec reted into the medium by recombinant L-form strains. The synthesis of PAC was growth-associated and stably regulated. Expression, secretion, and processing were not temperature-dependent and occurred at 26 degr ees C, 32 degrees C and even 37 degrees C. The expression vector pHC1 carried the pac gene under the control of the lac UV promotor and a ka namycin resistance gene. It could be maintained in L-form cells, showi ng low structural as well as segregational instability. The secretion of the biologically active enzyme into the medium indicated that the p ostranslational processing of the PAC molecule, including the excision of a 54 amino acid spacer peptide between the alpha and beta subunit, is not carried out in the periplasmic space, but occurs at the cytopl asmic membrane or autocatalytically.