Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase.
The 1923 bp cDNA for rat hepatic cholesteryl ester hydrolase (CEH) was cloned by screening a lambda gt11 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.
Ghosh
S
S
Department of Biochemistry, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-061, USA.
Mallonee
D H
DH
Hylemon
P B
PB
Grogan
W M
WM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers / Q000737:chemistry; D004950:Esterases / Q000235:genetics; D015723:Gene Library; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D002787:Sterol Esterase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013656:Taurocholic Acid / Q000494:pharmacology; D014162:Transfection
D000596:Amino Acids; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013656:Taurocholic Acid; D004950:Esterases; C022635:serine esterase; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase; D002787:Sterol Esterase