Expression and purification of recombinant human apolipoprotein A-I in Chinese hamster ovary cells.
The expression of apolipoprotein A-I (apoA-I) has been shown to be very difficult due to its amphiphilic character, autoaggregation, and degradation. We have expressed apoA-I using CHO cells, Baculovirus, and Escherichia coli [Schmidt et al., J. Biol. Chem. (1995) 270, 469-475]. Here we report about optimized conditions for the expression of proapoA-I in CHO cells, testing various serum-free media. We were able to yield apoA-I expression up to 80 micrograms/ml, by far the highest ever reported. However, immunoblot analysis revealed degraded apoA-I. The best apoA-I expression testing various conditions was about 20-30 micrograms/ml without any evidence of degradation. Interestingly, the apoA-I expression resulted in reproducible apoA-I fragments of 26 and 14 kDa. These fragments are consistent with already reported in vivo findings, in which carboxy-terminal proteolysis was suggested. The use of the protease inhibitors pepstatin and chymostatin, both carboxy-peptidase inhibitors, did result in contrast to other studied protease inhibitors in increased apoA-I yield. Therefore, limited carboxy-terminal proteolysis contributes to the degradation of CHO cell-secreted apoA-I. In addition, we evaluated various purification methods for the preparative isolation of recombinant apoA-I. In our hands we obtained the best recovery and no degradation with reversed-phase chromatography using a FPLC system.
Schmidt
H H
HH
Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Germany.
Genschel
J
J
Haas
R
R
Büttner
C
C
Manns
M P
MP
D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D016895:Culture Media, Serum-Free; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification*
D016632:Apolipoprotein A-I; D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins