Glycine-induced extracellular secretion of a recombinant cytochrome expressed in Escherichia coli.

Naheed Nazly Kaderbhai, Amna Karim, Wendy Hankey, Glyn Jenkins, Jamie Derek Venning, Mustak Ali Kaderbhai

Research output: Contribution to journalArticlepeer-review

37 Citations (SciVal)

Abstract

The effect of each of 20 different amino acid supplements to the growth medium of Escherichia coli on the extracellular release of a periplasmic recombinant cytochrome b 5 was investigated. Only glycine, and to a lesser extent histidine, stimulated the synthesis of secretory cytochrome b 5, as well as its discharge into the medium. Extracellular amounts of cytochrome b 5 accrued with increasing concentrations of exogenous glycine and duration of the culture period, in spite of the fact that increasing glycine in the medium progressively inhibited cell growth. For example, 1% medium glycine caused a 50% reduction in bacterial growth, but doubled the periplasmic pool of cytochrome b 5 to over 25 μg of cytochrome b 5/ml of culture at 24 h, a period during which almost all of cellular haemoprotein pool was turned over into the medium. A comparative study of the exportable form of cytochrome b 5 with a (non-secretory) cytoplasmic-resident counterpart indicated that the periplasmic cytochrome b 5 content was selectively discharged into the medium when less than 1% glycine was present, but, at higher doses, a significant proportion of the additional extracellular haemoprotein was derived from cell lysis. Optimal level of periplasmic discharge of the cytochrome required both active protein synthesis and the presence of a glycine supplement in the medium from the onset of bacterial growth. Phase-contrast and scanning electron microsocopy of glycine-grown Escherichia coli showed that the cells had a 3-7-fold enlarged 'eyeball' spheroidal morphology, with a condensed pericircular cytoplasm. The bulk of the volume in such hypertrophied cells consisted of the periplasm; this was reflected by the progressively lowered buoyancy of E. coli cultured with increasing amounts of glycine. The fragility of such cells was apparent by their marked sensitivity to lysis at glycine concentrations above 1%. We conclude that supplementation of E. coli cultures with moderate amounts of glycine substantially stimulates the synthesis of exportable proteins and further enhances their yield by discharge into the growth medium.

Original languageEnglish
Pages (from-to)53-61
Number of pages9
JournalBiotechnology and Applied Biochemistry
Volume25
Issue number1
DOIs
Publication statusPublished - Feb 1997

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