Transient expression of wild type and mutant human apolipoprotein AI in COS cells.
A human apolipoprotein AI (apo AI) minigene and two mutants were cloned into the vector pUHD10-1 for expression studies in COS cells under the control of the strong CMV (cytomegalovirus) enhancer and the own apo AI promoter. In the mutated apo AI minigene (mutant M1) the positions of the triplets of Gln(-2)-Gln-1 at the C-terminus of the prosequence were exchanged against Gln(-8)-Ala-7, the recognition site of the signal peptidase of the wild type human apo AI. The prosequence has been deleted in mutant M2 and the presequence linked directly to the N-terminus of the mature apo AI form. We report here on expression studies in COS cells, a cell line, which does not express apo AI. They were transfected by electroporation with pUHD10-1 constructs, which contain a) the wild type apo AI minigene and b) the two mutant apo AI minigenes with mutations described above. The following results were obtained: a) the wild type and mutant apo AI constructs were efficiently transcribed and translated in COS cells, b) the expression of the wild type preproapo AI minigene in COS cells led to the secretion of proapo AI (29 kDa), that of the mutant (M2) gene, devoid of the prosequence of mature apo AI (28.4 kDa), whereas the product of mutant gene M1 (31 kDa) with the recognition site of the signal peptides transposed to the C-terminus of the prosequence remained uncleaved within the COS cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Stoffel
W
W
Institut für Biochemie, Medizinische Fakultät der Universität zu Köln.
Binczek
E
E
D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000235:genetics* / Q000378:metabolism; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015870:Gene Expression; D005813:Genes, Synthetic / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D011487:Protein Conformation; D013462:Sulfur Radioisotopes; D014158:Transcription, Genetic / Q000235:genetics
D016632:Apolipoprotein A-I; D013462:Sulfur Radioisotopes