The cloning and overexpression of E. coli acyl carrier protein (ACP).

Acyl carrier protein (ACP) is a small (9kDa) protein which is essential for fatty acid synthesis. In bacteria [ l ] and plants [2] which use type I1 dissociable fatty acid synthetases, acyl chains are esterified to ACP through the 4'-phosphopantetheine prosthetic group, and ACP acts as a carrier during fatty acid synthesis. ACPs from bacteria and plants are very similar. All are small acidic proteins with conserved regions particularly around the serine residue through which the prosthetic group is attached. The similarity of E. coli ACP to many plant ACPs is such that E. coli ACP can be used as a substitute for plant ACP in many assays for plant fatty acid synthetase activity; indeed it sometimes proves more active in vitro than the native plant ACP [3]. This is useful because such large quantities of plant material are required to isolate relatively small amounts of protein. It is therefore easier to isolate E. coli ACP. E . coli contains only one ACP isoform and also has the advantage of being active in many plant system, whereas plant ACPs often do not work in other plant systems, and different and specific isoforms occur in different tissues [4]. It is possible to isolate 20-30mg of ACP from 200g of E. coli wet cell paste in 3 days. However, assaying plant fatty acid synthesis enzymes requires large quantities of ACP and. therefore,