Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase

• Published in: Biochimica et biophysica acta Vol. 1259; no. 3; pp. 305 - 312
• Main Authors: Ghosh, Shobha, Mallonee, Darrell H., Hylemon, Philip B., Grogan, W.McLean
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 07.12.1995
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Animals; Base Sequence; Binding Sites; Blotting, Northern; Blotting, Western; Carboxylesterase; Carboxylic Ester Hydrolases - genetics; cDNA; Cholesteryl ester hydrolase; Cloning; Cloning, Molecular; Conserved Sequence; DNA Primers - chemistry; Esterases - genetics; Gene Library; Liver - enzymology; Molecular Sequence Data; Pancreas - enzymology; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; Sterol Esterase - chemistry; Sterol Esterase - genetics; Sterol Esterase - metabolism; Taurocholic Acid - pharmacology; Transfection; Cholesteryl ester hydrolase; SDS-PAGE; Cloning; rRNA; CEH; IPTG; cDNA; mRNA; Carboxylesterase; CMV; PCR
• ISSN: 0005-2760; 0006-3002; 1879-145X
• DOI: 10.1016/0005-2760(95)00184-0

The 1923 bp cDNA for rat hepatic cholesteryl ester hydrolase (CEH) was cloned by screening a λgt11 expression library with an oligonucleotide containing the consensus active site sequence for cholesteryl esterases. Expression of a fusion protein, cross-reacting with antibody to the purified liver CEH, was demonstrated by Western blot analysis. The cDNA was sequenced and found to have only 44% homology with pancreatic CEH. Although unique, the cDNA sequence exhibited much greater overall homology with liver carboxylesterases, in both coding and 5′/3′ non-coding regions. In Northern blot analysis, the cDNA hybridized with a single band from liver mRNA but not with pancreatic mRNA. The 1.7 kb coding sequence, predicting a 62 kDa protein, was cloned into an Escherichia coli expression system with an inducible promoter and into COS-7 cells. Both expression systems produced a protein which comigrated with liver CEH (66 kDa) on SDS-PAGE and immunoreacted with antibodies to liver CEH on Western blots. Whereas the prokaryotic system produced an inactive protein, expression in COS-7 cells was accompanied by a 5-fold increase in CEH activity and a corresponding increase in immunoreactive protein.