LACTOSE FED-BATCH OVEREXPRESSION OF RECOMBINANT METALLOPROTEINS IN ESCHERICHIA-COLI BL21(DE3) - PROCESS-CONTROL YIELDING HIGH-LEVELS OF METAL-INCORPORATED, SOLUBLE-PROTEIN

A method for producing recombinant proteins in pilot scale fermentatio n equipment using a glucose fed-batch initial growth, followed by a mi dlog phase feeding of a glucose and lactose mixture is described. Usin g the host strain Escherichia coli BL21(DE3), the diiron protein stear oyl-acyl carrier protein Delta(9) desaturase has been overexpressed at a biomass level of up to 12 g x liter(-1) dry cell weight, representi ng a 12-fold increase in volumetric productivity relative to that obta ined from batch fermentations. Under these conditions, a maximum of 36 % of the total cellular protein accumulates as the desaturase polypept ide. A correlation between the slowed growth rate of the fed-batch cul ture, a continued, albeit slower, exponential growth under inducing co nditions, and a favorable partitioning between formation of the solubl e holoprotein and inclusion bodies is reported. This correlation sugge sts that fed-batch techniques can be used to beneficially influence ra te-limiting processes in the maturation of overexpressed proteins, suc h as metal uptake and incorporation proposed here. By using cells prod uced from the fed-batch method, the iron-containing, soluble desaturas e can be purified in a yield of up to 66 mg x g(-1) dry cell weight (s imilar to 500 mg X liter(-1) culture), representing a three to fivefol d increase in the yield relative to that obtained from batch fermentat ions. In addition, these methods are suitable for the production of th e Anabena 7120 vegetative [2Fe 2S] ferredoxin in E. coli BL21(DE3) pLy sS, a host strain used for the overexpression of toxic proteins. (C) 1 995 Academic Press, Inc.