cDNA cloning and expression of human complement component C2.
A full-length cDNA clone for C component C2 was isolated from a human liver cDNA library in lambda gt11 by initially screening with an affinity-purified rabbit anti-C2 antibody and then using the isolated partial C2 cDNA as a probe for re-screening the library. The cDNA insert of clone lambda C2HL5-3 was sequenced in its entirety. It consisted of 2961 nucleotides including a 5' untranslated region of 388 nucleotides, followed by a 60 nucleotide region coding for a putative signal peptide, a 2196 nucleotide region coding for the 732 amino acids of the mature C2 polypeptide, and a 317 nucleotide long 3' untranslated region. Comparison of the nucleotide sequence to the previously reported C2 cDNA sequence showed two major differences. First, the 5' untranslated region of C2HL5-3 was 352 nucleotides longer and included four ATG followed by in-frame termination codons. Second, nucleotide residue 1987 was a C instead of a G, resulting in a change of amino acid residue 513 from Leu to Phe and in the appearance of an EcoRI site. The full-length C2 cDNA was cloned into the expression vector p91023(B). Transfection of the recombinant plasmid in COS cells resulted in the secretion of a protein with antigenicity and hemolytic activity similar to those of native C2. Western blot analysis indicated that secreted rC2 had the same apparent m.w. as C2 in human serum. Northern blot analysis of total RNA isolated from transfected COS cells showed two bands of C2 mRNA, both of which were longer than human liver C2 mRNA and represent transcripts generated by the vector-C2 construct.
Horiuchi
T
T
Department of Medicine, University of Alabama, Birmingham 35294.
Macon
K J
KJ
Kidd
V J
VJ
Volanakis
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003175:Complement C2 / Q000235:genetics* / Q000302:isolation & purification; D004247:DNA / Q000302:isolation & purification*; D000882:Haplorhini; D006801:Humans; D008969:Molecular Sequence Data; D014162:Transfection
D003175:Complement C2; D004247:DNA