PMID: 8962082Dec 10, 1996Paper

Cloning and expression of the multifunctional human fatty acid synthase and its subdomains in Escherichia coli

Proceedings of the National Academy of Sciences of the United States of America
A JayakumarS J Wakil

Abstract

We engineered a full-length (8.3-kbp) cDNA coding for fatty acid synthase (FAS; EC 2.3.1.85) from the human brain FAS cDNA clones we characterized previously. In the process of accomplishing this task, we developed a novel PCR procedure, recombinant PCR, which is very useful in joining two overlapping DNA fragments that do not have a common or unique restriction site. The full-length cDNA was cloned in pMAL-c2 for heterologous expression in Escherichia coli as a maltose-binding protein fusion. The recombinant protein was purified by using amylose-resin affinity and hydroxylapatite chromatography. As expected from the coding 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-human FAS (MBP-hFAS) catalyzed palmitate synthesis from acetyl-CoA, malonyl-CoA, and NADPH and exhibited all of the partial activities of FAS at levels comparable with those of the native human enzyme purified from HepG2 cells. Like the native HepG2 FAS, the products of MBP-hFAS are mainly palmitic acid (> 90%) and minimal amounts of stearic and arachidic acids. Similarly, a human FAS cDNA encoding domain ...Continue Reading

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Citations

Aug 2, 2007·Origins of Life and Evolution of the Biosphere : the Journal of the International Society for the Study of the Origin of Life·Giovanni Murtas
Apr 12, 2003·Progress in Lipid Research·Stuart SmithAnil K Joshi
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Mar 15, 2001·Proceedings of the National Academy of Sciences of the United States of America·S S ChiralaS J Wakil
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