Production of mature human apolipoprotein A-I in a baculovirus-insect cell system: propeptide is not essential for intracellular processing but may assist rapid secretion.
To achieve expression of human mature apolipoprotein A-I (apoA-I) in the baculovirus-insect cell expression system, the propeptide encoding region of full-length preproapoA-I was deleted using polymerase chain reaction and the resulting cDNA was cloned into BacPak8 plasmid. After transfection into Sf21 insect cells and plaque purification, mature human apoA-I was secreted by the infected cells into the medium as determined by immunoblotting, amino-terminal sequencing, and molecular weight determination. In both monolayer cell cultures, and in suspension cell culture, maximum expression was achieved by the fifth day. For the first 4 days, 50 to 70% of the synthesized apoA-I was retained in the cells. This intracellular apoA-I was represented by mature apoA-I as shown by immunoblotting and amino-terminal sequencing. Further incubation resulted in a sharp decrease in the cell apoA-I content without a corresponding increase in protein in the medium and most likely represents intracellular degradation of the protein. We conclude that the deletion of the propeptide, while not preventing the correct cleavage of prepeptide during intracellular processing, results in reduced secretion of mature apoA-I. The baculovirus-insect cell expression system described in this study provides a useful method for producing recombinant mature apoA-I and is a potential tool for understanding the function of propeptide in intracellular transport and secretion of apoA-I from cells.
Pyle
L E
LE
Baker Medical Research Institute, Prahran, Victoria, 3181, Australia.
Fidge
N H
NH
Barton
P A
PA
Luong
A
A
Sviridov
D
D
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000235:genetics*
D016632:Apolipoprotein A-I; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins