Purification and characterization of recombinant hamster tissue complement C1s.
Hamster complement C1s cDNA was inserted into expression plasmid BCMGSNeo, and transfected to SEA7 cells, A31 mouse fibroblasts transformed by polyoma virus. The transfectant secreted a large amount of recombinant C1s that was activated in the serum free culture medium and hydrolyzed acetyl-Gly-L-Lys-naphthyl ester (AGLNE). C1s was purified to a homogeneity from the culture medium of the transfectant by DEAE-Sephadex, Dymatrex orange A and size-exclusion HPLC. Purified hamster C1s consumed human complement in hemolytic assay and hydrolyzed gelatin in enzymography. To investigate the enzymic action of C1s at molecular levels, several antibodies were prepared against hamster C1s. One peptide (amino-acid residues 379-391) and two peptides (amino-acid residues 478-496 and 560-583) corresponding to the heavy and the light chain, respectively, were synthesized. The amino-acid sequences of these regions is not conserved between hamster and human C1s. Antibodies against these peptides were raised in rabbits. The anti-peptide antibodies bound specifically to hamster serum and recombinant C1s but not to human C1s. They inhibited the esterase activity of recombinant C1s to varying degrees depending on each antibody's binding site.
Toyoguchi
T
T
Division of Physiology and Pathology, National Institute of Radiological Science, Chiba, Japan.
Yamaguchi
K
K
Imajoh-Ohmi
S
S
Kato
N
N
Kusunoki
M
M
Kageyama
H
H
Sakiyama
S
S
Nagasawa
S
S
Moriya
H
H
Sakiyama
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D003173:Complement C1s / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D015941:Complement Hemolytic Activity Assay; D006224:Cricetinae; D005780:Gelatin / Q000737:chemistry; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification; D014162:Transfection
D000906:Antibodies; D009842:Oligopeptides; D011994:Recombinant Proteins; D005780:Gelatin; D003173:Complement C1s