Abstract
Recently, Bora et al. (Bora, P. S., Bora, N. S., Wu, X., and Lange, L. G. (1991) J. Biol. Chem. 266, 16774-16777) reported the cloning and expression of a human fatty acid ethyl ester synthase III (FAEES-III) cDNA that has only four amino acid substitutions compared with human glutathione S-transferase (GST) GSTP1-1, and, when expressed in MCF-7 cells, the protein has both FAEES and GST activities. By site-directed mutagenesis of a GSTP1 cDNA, we have constructed a clone that encodes the FAEES-III protein described by Bora et al. (1991). The recombinant FAEES-III protein was expressed in Escherichia coli and has been shown to be devoid of FAEES and GST activities. The recombinant FAEES-III protein does not bind to a glutathione agarose affinity matrix, presumably because two of the substituted amino acids, Trp-39-->Cys and Gln-52-->Glu, are thought to contribute to the GST glutathione binding site. One of the base substitutions in the FAEES-III cDNA encodes an extra SacI site not found in the GSTPI cDNA. Polymerase chain reaction amplification of human genomic DNA has identified the GSTPI gene, but no DNA from the proposed FAEES gene with a diagnostic SacI site has been detected. Evaluation of the hybridization pattern of HindIII genomic restriction fragments has identified fragments that contain the GSTPI gene and a pseudogene (Board et al. 1992), and there do not appear to be any hybridizing fragments that could contain the FAEES-III gene. Our results do not provide any evidence in support of a relationship between FAEES-III and GST, and the cDNA reported by Bora et al. (1991) may have resulted from a cloning artifact.
Highlights
From the Molecular Genetics Group,John Curtin School of Medical Research,Australian National University, Canberra, Australian Capital Territory 2601, Australia
The amino acid sequence of GSTPl has been deduced from cDNA and genomic clones (Kano et al, 1987; Board et al 1989; Cowelel t al., 1988;Moscow et al 1988),and the complete binant FAEES-I11protein does not bind to a glutathi- sequence has been determined by peptide sequencing (Ahmad one agarose affinity matrix, presumably because two et al, 1990).Recent studies by Suzuki et al (1990)and Sharma of the substitutedamino acids, Trp-39 -.,Cys and Gln- et al (1991) have not been able to find any FAEES activity
The PCR reactionwas carried out ina Corbett cDNA encoding theGSTP lprotein was cloned downstream
Summary
Expression of GSTPl and FAEES-111 cDNAs in E. coli-A out a t 72 "C for 1min. The resulting clone pGT3EX expressed GSTP1-1protein from GSTP1-1 purified from placenta (data not shown). A clonethat expresses the FAEES-111protein described by Bora etal. Purification of GSTPl-1 or FAEES-111 Protein-The. GSTP1-1protein expressedby pGT3EX could be readily purified by affinity chromatography on glutathione agarose as described bySimmons and VandeJragt (1977). Was detected by polyclonal rabbit antibodies to GSTP1-1 and a goat the FAEES-I11 protein expressed by pGT3FA did not bind to anti-rabbit IgG alkaline phosphatase conjugate (Sigma) by a method described in detail by Board (1984). These primers are selected from areas that ariedentical in the GSTPl FoAr EES-111cDNA sequences.
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