Expression of polypeptide segments of the human complement component C3 in E. coli: genetic and immunological characterization of cDNA clones specific for the alpha-chain of C3.
The third component of complement C3 and its fragments have a central role in a variety of host defense mechanisms. The identification of functionally relevant C3 domains is important because of the marked functional versatility of the C3 molecule. Several human C3 cDNA clones from a human liver cDNA library were isolated and characterized. A bacterial expression vector system was used to express cDNA clones that were identified by an immunological screening procedure. The C3 cDNA clones produced in E. coli the hybrid proteins consisting of cro-beta-galactosidase and polypeptide segments of human C3, as revealed by Western blotting with antisera to human C3. The C3 moiety of the hybrid proteins had a m.w. of up to 46.000. Polyclonal antibodies against the C3 segments expressed by one of the C3 cDNA clones (ReC3-1) have been raised in mice and rabbit, and in addition, a monoclonal antibody was produced. The antisera and the monoclonal antibody reacted in Western blotting analysis selectively with the alpha-chain, but not the beta-chain of human C3. Restriction mapping of the different cDNA clones was performed, and revealed that the different clones were partially overlapping. The ReC3-1 cDNA clone included a 0.7 kb noncoding region at the 3' terminal end of the C3 cDNA. One of the restriction sites (Hind III) identified in the ReC3-1 cDNA clone was not present in the recently published sequence of human C3 cDNA. This difference in nucleotide sequence provides direct evidence for C3 polymorphism at the DNA level. The combination of immunologic procedures with recombinant DNA methodology should facilitate additional analysis of the structure-function relationship of the C3 molecule.
D000818:Animals; D003001:Cloning, Molecular; D003176:Complement C3 / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008099:Liver; D051379:Mice; D010446:Peptide Fragments / Q000302:isolation & purification*; D011817:Rabbits
D003176:Complement C3; D004269:DNA, Bacterial; D010446:Peptide Fragments; D004247:DNA; D004262:DNA Restriction Enzymes