The expression of hemolytically active human complement protein C9 in mammalian, insect, and yeast cells.
The cDNA sequence encoding mature human C9 protein and its signal peptide was cloned into three expression vectors for expression in COS-7 (mammalian), Spodoptera frugiperda IPLB-SF-21AE (insect), and Saccharomyces cerevisiae (yeast) cells. In addition, C9 cDNA encoding only the mature protein was fused to the yeast invertase leader sequence (SUC2) and cloned for expression in yeast. Under optimal conditions COS-7 and IPLB-SF-21AE cells secreted recombinant C9 (rC9) at concentrations of about 111 and 700 ng C9/ml culture supernatant, respectively. By comparison S. cerevisiae, whether transformed with C9 cDNA containing its native or yeast invertase leader sequence, secreted only very small amounts of rC9 (5-10 ng/ml). However, upon lysis concentrations of up to 500 ng/mg dry wt were found in yeast cells transformed with C9 cDNA. SDS-PAGE followed by Western blot analysis revealed COS-7 cell and S. cerevisiae expressed rC9 to have a MW similar to that of native C9 purified from human serum, while rC9 from IPLB-SF-21AE cells was about 4 kDa smaller. No hemolytic activity of S. cerevisiae secreted rC9 could be detected and the specific hemolytic activity of S. cerevisiae intracellular rC9 was also very low. However, the specific hemolytic activities of COS-7 and IPLB-SF-21AE secreted rC9 were indistinguishable from that of purified native human C9. Thus, for future studies on the structure and function of C9 where the production of large quantities of mutant protein would be desirable, the baculovirus-insect cell expression system appears to offer considerable advantages.
Tomlinson
S
S
Department of Comparative and Experimental Pathology, University of Florida, Gainesville 32610.
Ueda
E
E
Maruniak
J E
JE
Garcia-Canedo
A
A
Bjes
E S
ES
Esser
A F
AF
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D002522:Chlorocebus aethiops / Q000235:genetics; D003001:Cloning, Molecular; D003186:Complement C9 / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D009036:Moths; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection; D043324:beta-Fructofuranosidase
D003186:Complement C9; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase