CLONING AND EXPRESSION OF THE MULTIFUNCTIONAL HUMAN FATTY-ACID SYNTHASE AND ITS SUBDOMAINS IN ESCHERICHIA-COLI

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synth ase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we charact erized previously. In the process of accomplishing this task, we devel oped a novel PCR procedure, recombinant PCR, which is very useful in j oining two overlapping DNA fragments that do not have a common or uniq ue restriction site. The full-length cDNA was cloned in pMAL-c2 for he terologous expression in Escherichia coli as a maltose-binding protein fusion. The recombinant protein was purified by using amylose-resin a ffinity and hydroxylapatite chromatography. As expected from the codin g capacity of the cDNA expressed, the chimeric recombinant protein has a molecular weight of 310,000 and reacts with antibodies against both human FAS and maltose-binding protein. The maltose-binding protein-hu man FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malo nyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified fr om HepG2 cells. Like the native HepG2 FAS, the products of MBP-hFAS ar e mainly palmitic acid (>90%) and minimal amounts of stearic and arach idic acids. Similarly, a human FAS cDNA encoding domain I (beta-ketoac yl synthase, acetyl-CoA and malonyl-CoA transacylases, and beta-hydrox yacyl dehydratase) was cloned and expressed in E. coli using pMAL-c2. The expressed fusion protein, MBP-hFAS domain I, was purified to appar ent homogeneity (M(r) 190,000) and exhibited the activities of the ace tyl/malonyl transacylases and the beta-hydroxyacyl dehydratase. In add ition, a human FAS cDNA encoding domains II and III (enoyl and beta-ke toacyl reductases, acyl carrier protein, and thioesterase) was cloned in pET-32b(+) and expressed in E. coli as a fusion protein with thiore doxin and six in-frame histidine residues. The recombinant fusion prot ein, thioredoxin-human FAS domains II and III, that was purified from E. coli had a molecular weight of 159,000 and exhibited the activities of the enoyl and beta-ketoacyl reductases and the thioesterase. Both the MBP and the thioredoxin-His-tags do not appear to interfere with t he catalytic activity of human FAS or its partial activities.