Expression and partial purification of a recombinant secretory form of human liver carboxylesterase.
Serine-dependent carboxylesterases (EC 3.1.1.1) are found in a variety of tissues with high activity detected in the liver. Carboxylesterases (CaE) hydrolyze aliphatic and aromatic esters, and aromatic amides, and play an important role in the detoxification of xenobiotic chemicals that contain organophosphate (OP) compounds. The detoxifying ability of CaE is limited by its low concentration in serum where it encounters OP compounds. Studies in our laboratory have shown that a pRC/CMV-hCaE plasmid construct, stably integrated into 293T cells, expresses a human liver CaE in culture. However, the enzyme remained inside the cell and reached a low steady-state level of expression. The goals of this study were to overexpress a functional human liver CaE from a recombinant cDNA in a human cell line and to isolate and purify the recombinant protein. To accomplish these goals, a single amino acid change was made in the C-terminal retrieval signal, HIEL (His-Ile-Glu-Leu), of human liver CaE. The mutation produced a unique Eco47III restriction site, which aided in clone selection. The recombinant plasmid, pRc/CMV-mhCaE, was isolated and stably integrated into human 293T cells. Expression of the altered cDNA resulted in secretion of an active CaE up to levels of 500 enzyme units per liter of growth medium. Secretory CaE displayed isoelectric focusing patterns similar to those of the native enzyme with no observable changes in activity. The secreted enzyme was partially purified by hydrophobic interaction chromatography and Cibacron blue affinity chromatography. Partial enzyme purification was achieved, and CaE retained a high level of enzymatic activity.
Scott
D F
DF
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia 30912, USA.
Chacko
T L
TL
Maxwell
D M
DM
Schlager
J J
JJ
Lanclos
K D
KD
D001483:Base Sequence; D015153:Blotting, Western; D043182:Carboxylesterase; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; D043182:Carboxylesterase