Abstract
The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26°C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotrophic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.
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References
Bowden GA, Georgiou G (1990) Folding and aggregation of β-lactamase in the periplasmic space of Escherichia coli. J Biol Chem 265:16760–16766
Bowden GA, Paredes AM, Georgiou G (1991) Structure and morphology of protein inclusion bodies in Escherichia coli. Bio/Technology 9:725–730
Bruns W, Hoppe J, Tsai H, Brünning HJ, Maywald F, Collins J, Mayer H (1985) Structure of penicillin acylase gene of E. coli:a periplasmic enzyme that undergoes multiple proteolytic processing. J Mol Appl Genet 3:36–44
Chalmers JJ, Kim E, Telford JN, Wong EY, Tacon WC, Shuler ME, Wilson DB (1990) Effects of temperature on Escherichia coli overproducing β-lactamase or human epidermal growth factor. Appl Environ Microbiol 56:104–111
Choi KS, Kim JA, Kang HS (1992) Effects of site-directed mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC11105. J Bacteriol 174:6270–6278
Ferdinand W (1964) The isolation and specific activity of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. Biochem J 92:578–585
Georgiou G, Telford JN, Shuler ML, Wilson DB (1986) Localization of inclusion bodies in Escherichia coli overproducing β-lactamase or alkaline phosphatase. Appl Environ Microbiol 52:1157–1161
Haase-Pettingell CA, King J (1988) Formation of aggregates from a thermolabile in vivo folding intermediate in P22 tailspike maturation. A model for inclusion body formation. J Biol Chem 263:4977–4983
Hunt PD, Tolley SP, Ward RJ, Hill CP, Dodson GG (1990) Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC11105. Protein Eng 3:635–639
Kopetzki E, Schumacher G, Buckel P (1989) Control of formation of active soluble or inactive insoluble baker's yeast α-glucosidase PI in Escherichia coli by induction and growth conditions. Mol Gen Genet 216:149–155
Kutzbach C, Rauenbusch E (1974) Preparation and general properties of crystalline penicillin acylase from Escherichia coli ATCC11105. Z Phys Chem 355:45–53
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227:680–685
McIver K, Kessler E, Ohman DE (1991) Pseudomonas aeroginosa lasB1 mutants produce an elastase, substituted at active-site His-223, that is defective in activity, processing and secretion. J Bacteriol 173:7781–7789
Mizukami T, Komatsu Y, Hosoi N, Itoh S, Oka T (1986) Production of active human interferon-β in E. coli. I. Preferential production by lower culture temperature. Biotechnol Lett 8:605–610
Oliver G, Valle F, Rosetti F, Gomez-Pedroso M, Santamaria P, Gosset G, Bolivar F (1985) A common precursor for the two subunits of the penicillin acylase from E. coli ATC 11105. Gene 40:9–14
Robas N, Branlant C (1994) The expression of the penicillin G amidase gene of E. coli by primer extension analysis. Curr Microbiol (in press)
Robas N, Zouheiry H, Branlant G, Branlant C (1993) Improved penicillin amidase production using a genetically engineered mutant of Escherichia coli ATCC11105. Biotechnol Bioeng 41:14–24
Schein CH (1990) Solubility as a function of protein structure and solvent component. Bio/Technology 8:308–312
Schumacher G, Sizmann D, Hang H, Bückel P, Böck A (1986) Penicillin amylase from Escherichia coli: a unique gene-protein relation. Nucleic Acids Res 14:5713–5725
Schnaitman CA (1971) Effect of ethylenediaminetetraacetic acid, Triton X100, and lysozyme on the morphology and chemical composition of isolated cell walls of Escherichia coli. J Bacteriol 108:545–552
waley SG (1974) A spectrophotometric assay of β-lactamase action on penicillins. Biochem J 139:789–790
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Scherrer, S., Robas, N., Zouheiry, H. et al. Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli Penicillin G amidase precursor polypeptide. Appl Microbiol Biotechnol 42, 85–91 (1994). https://doi.org/10.1007/BF00170229
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DOI: https://doi.org/10.1007/BF00170229