Synthesis and secretion of recombinant penicillin G acylase in bacterial L-forms.
L-form strains of Proteus mirabilis and Escherichia coli lacking the cell wall represent an alternative prokaryotic cell system for the production of recombinant proteins (KLESSEN et al. 1988, LAPLACE et al. 1988a, 1989b). We could demonstrate that they are also able to synthesize the enzyme penicillin G acylase (PAC)1). PAC was processed and secreted 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 degrees 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 kanamycin resistance gene. It could be maintained in L-form cells, showing low structural as well as segregational instability. The secretion of the biologically active enzyme into the medium indicated that the postranslational 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 cytoplasmic membrane or autocatalytically.
Gumpert
J
J
Institut für Molekulare Biotechnologie e. V., Jena, Germany.
Cron
H
H
Plapp
R
R
Niersbach
H
H
Hoischen
C
C
D002473:Cell Wall; D003470:Culture Media; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D010399:Penicillin Amidase / Q000096:biosynthesis* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011513:Proteus mirabilis / Q000201:enzymology / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism; D013696:Temperature; D014169:Transformation, Bacterial
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D010399:Penicillin Amidase