AN ACYL-CARRIER-PROTEIN-THIOESTERASE DOMAIN FROM THE 6-DEOXYERYTHRONOLIDE-B SYNTHASE OF SACCHAROPOLYSPORA-ERYTHRAEA - HIGH-LEVEL PRODUCTION, PURIFICATION AND CHARACTERIZATION IN ESCHERICHIA-COLI

作者：CAFFREY P; GREEN B; PACKMAN LC; RAWLINGS BJ; STAUNTON J; LEADLAY PF

来源：European Journal of Biochemistry, 1991, 195(3): 823-830.

DOI：10.1111/j.1432-1033.1991.tb15771.x

The C-terminal region of a multifunctional polypeptide from the 6-deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176-178]. Site-directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7-based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post-translationally modified by attachment of a 4'-phosphopantethine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.

• 出版日期1991-2-14