Expression of a Gossypium hirsutum cDNA encoding a FatB palmitoyl-acyl carrier protein thioesterase in Escherichia coli
2002
Huynh, T.T. ((University of North Texas, Denton (Etats Unis). Department of Biological Sciences)) | Pirtle, R.M. | Chapman, K.D.
A cotton FatB cDNA encoding a palmitoyl-acyl carrier protein (ACP) thioesterase (Genbank Accession number AF034266) was expressed in various Escherichia coli strains. Transcription and translation in a coupled in vitro system revealed the presence of two [35S-Met]-labeled protein products, one of about 35 kDa and one of about 46 kDa. The 46 kDa polypeptide likely represented the translation of the preprotein while the 35 kDa polypeptide likely represented a translation product initiated at an alternative, internal in-frame initiation codon. Polyclonal anti-peptide antibodies were used to confirm the accumulation of this truncated protein. An immunoreactive 35 kDa protein was recognized in transformed E. coli cell lysates supporting the notion that indeed there was an internal start site, which seemed to be preferred when the cotton cDNA was expressed in E. coli. In crude homogenates of cotton embryos (30 dpa, days post anthesis) and cotyledons of 48 h dark-grown seedlings a 37 kDa protein, which likely represents the mature processed FatB protein, was recognized. When acyl-CoA synthetase-minus E. coli mutants (K27 fadD88 mutant, CGSC 5478) were transformed with the cotton FatB cDNA, a four- to five-fold increase in C16:0 free fatty acid content was measured in the culture medium. Acyl-ACP thioesterase activity assays in E. coli lysates revealed that there was a clear preference for palmitoyl-ACP over oleoyl-ACP in vitro. Collectively, our results indicate that indeed the cotton FatB cDNA encodes a functional thioesterase with a preference for saturated acyl-ACPs (FatB) over unsaturated acyl-ACPs (FatA)
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