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 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.
Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705, USA.
B J
BJ
Hoffman
J A
JA
Broadwater
P
P
Johnson
J
J
Harper
B G
BG
Fox
W R
WR
Kenealy
D002455:Cell Division; D002478:Cells, Cultured; D003470:Culture Media; D004926:Escherichia coli / Q000166:cytology / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007785:Lactose / Q000378:metabolism*; D010865:Pilot Projects; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D013230:Stearoyl-CoA Desaturase / Q000096:biosynthesis* / Q000302:isolation & purification; D013997:Time Factors
D003470:Culture Media; D011994:Recombinant Proteins; D007501:Iron; D013230:Stearoyl-CoA Desaturase; D005947:Glucose; D007785:Lactose