Overexpression and purification of the Escherichia coli inner membrane enzyme acyl-acyl carrier protein synthase in an active form.
Acyl-acyl carrier protein synthase (Aas) is widely used to synthesize thioester adducts of fatty acids between 8 and 18 carbons in length enzymatically to the phosphopantetheine group of acyl carrier protein. The enzyme is an 80.6-kDa inner membrane protein that functions in vivo as a 2-acylglycerophosphoethanolamine acyltransferase. The E. coli aas open reading frame was inserted into the expression plasmid pET28a so that, upon expression, a 21-amino-acid extension containing 6 consecutive histidine residues was added to the carboxyl terminus. The plasmid was designated pAasH. The activity of Aas in membranes was assessed from several cell lines. Membranes from the commonly used host line BL21(DE3) containing pAasH accumulated 30-fold and 38-fold more Aas activity than membranes from BL21(DE3) cells lacking the plasmid, when induced with isopropyl beta-d-thiogalactopyranoside (IPTG) or lactose, respectively. When pAasH was expressed under IPTG induction in cell line C41(DE3), a previously described cell line selected to enhance the expression of membrane proteins, Aas levels accumulated to 135-fold higher levels than in the cell line lacking the plasmid. Functional Aas can be isolated from either BL21(DE3) or C41(DE3) cell lines by differential centrifugation, followed by detergent extraction with Triton X-100 and nickel nitrilotriacetic acid affinity chromatography. The overexpression of Aas in cell line C41(DE3) is noteworthy compared to cell line BL21(DE3) because it results in a 3- to 4-fold higher accumulation of active enzyme in the membrane fraction and a lower proportion of inactive protein in the inclusion body.
Shanklin
J
J
Department of Biology, Brookhaven National Laboratory, Upton, New York, 11786, USA. shanklin@bnl.gov
D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase