PMID:8535158

Citation	Hoffman, BJ, Broadwater, JA, Johnson, P, Harper, J, Fox, BG and Kenealy, WR (1995) Lactose fed-batch overexpression of recombinant metalloproteins in Escherichia coli BL21 (DE3): process control yielding high levels of metal-incorporated, soluble protein. Protein Expr. Purif. 6:646-54
Abstract	A method for producing recombinant proteins in pilot scale fermentation equipment using a glucose fed-batch initial growth, followed by a midlog phase feeding of a glucose and lactose mixture is described. Using the host strain Escherichia coli BL21(DE3), the diiron protein stearoyl-acyl carrier protein delta 9 desaturase has been overexpressed at a biomass level of up to 12 g x liter-1 dry cell weight, representing a 12-fold increase in volumetric productivity relative to that obtained from batch fermentations. Under these conditions, a maximum of 36% of the total cellular protein accumulates as the desaturase polypeptide. A correlation between the slowed growth rate of the fed-batch culture, a continued, albeit slower, exponential growth under inducing conditions, and a favorable partitioning between formation of the soluble holoprotein and inclusion bodies is reported. This correlation suggests that fed-batch techniques can be used to beneficially influence rate-limiting processes in the maturation of overexpressed proteins, such as metal uptake and incorporation proposed here. By using cells produced from the fed-batch method, the iron-containing, soluble desaturase can be purified in a yield of up to 66 mg x g-1 dry cell weight (approximately 500 mg x liter-1 culture), representing a three to fivefold increase in the yield relative to that obtained from batch fermentations. In addition, these methods are suitable for the production of the Anabena 7120 vegetative [2Fe 2S] ferredoxin in E. coli BL21(DE3) pLysS, a host strain used for the overexpression of toxic proteins.
Links	PubMed Online version:10.1006/prep.1995.1085
Keywords	Cell Division; Cells, Cultured; Culture Media; Escherichia coli/cytology; Escherichia coli/metabolism; Fermentation; Glucose/chemistry; Glucose/metabolism; Iron/metabolism; Lactose/metabolism; Pilot Projects; Plasmids; Recombinant Proteins/biosynthesis; Stearoyl-CoA Desaturase/biosynthesis; Stearoyl-CoA Desaturase/isolation & purification; Time Factors
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