In vivo kinetics as a sensitive method for testing physiologically intact human recombinant apolipoprotein A-I: comparison of three different expression systems.
In order to assess the structural and functional integrity of recombinant human apoA-I, we expressed apoA-I using three different expression systems: Baculovirus transfected Spodoptera frugiperda (Sf9) cells, stably transfected Chinese hamster ovary (CHO) cells, and transformed Escherichia coli (E. coli). Purified apoA-I from the three expression systems was radioiodinated and their catabolism was compared in normolipemic rabbits. The kinetic turnover studies of radiolabelled apoA-I in normolipemic rabbits revealed that highly purified recombinant apoA-I had an identical decay curve compared to native apoA-I, regardless whether it was purified from Sf9 cells, CHO cells, or E. coli. We also determined the association of the three recombinant apoA-I forms with both rabbit and human HDL. All three recombinant apoA-I forms were associated with HDL2 and HDL3 after injection into the rabbits and after incubation with human serum using both a Superose 6 column separation system and density gradient ultracentrifugation. The addition of the pro-segment or the addition of methionine at the amino-terminal end of apoA-I did not alter its metabolism and association to HDL. In conclusion, all studied expression systems are capable of producing high levels of physiologically intact recombinant human apoA-I. The aminoterminal addition of the prosegment of apoA-I or methionine did not alter the in vivo metabolism of apoA-I or its association to HDL.
Schmidt
H H
HH
Department of Gastroenterology and Hepatology, Medizinische Hochschule Hannover, Germany.
Haas
R E
RE
Remaley
A
A
Genschel
J
J
Strassburg
C P
CP
Büttner
C
C
Manns
M P
MP
D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008297:Male; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D018411:Spodoptera
D016632:Apolipoprotein A-I; D011994:Recombinant Proteins