Expression of recombinant human apolipoprotein A-I in Chinese hamster ovary cells and Escherichia coli.
Human recombinant apolipoprotein (apo) A-I was produced by Chinese hamster ovary (CHO) cells and Escherichia coli with expression vectors containing cDNAs encoding preproapoA-I or apoA-I, respectively. The apoA-I from CHO cells was purified from the culture medium by ammonium sulfate precipitation, phenyl-Sepharose chromatography, and affinity purification on anti-apoA-I immunoabsorber. Human apoA-I was produced in E. coli as a fusion protein with glutathione S-transferase. A four amino acid linker, which separated the two proteins, was specifically recognized and cut by Factor Xa. The purification was accomplished by chromatography of E. coli extracts on glutathione-Sepharose and digestion with Factor Xa. The highest production level was found to be 0.5 micrograms/ml of culture medium per 48 h for a clone of stable transformant of CHO cells, whereas E. coli could produce as much as 20 micrograms/ml of bacterial culture. These apoA-I forms were compared in terms of molecular weight, isoelectric point, and expression of several epitopes. Recombinant apoA-I obtained from CHO cells appears intact and its isoelectric point is compatible with that of the mature form and the proform of apoA-I, whereas a part of the apoA-I produced by E. coli does not contain the COOH-terminus. Also, two of six epitopes are expressed to a greater extent in apoA-I obtained from E. coli than in apoA-I obtained from human plasma.
Brissette
L
L
Laboratoire du Métabolisme des Lipoprotéines, Institut de Recherches Cliniques de Montréal, Québec, Canada.
Cahuzac-Bec
N
N
Desforges
M
M
Bec
J L
JL
Marcel
Y L
YL
Rassart
E
E
D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D016632:Apolipoprotein A-I; D011994:Recombinant Proteins; D004247:DNA