Expression of hemolytically active human complement component C1r proenzyme in insect cells using a baculovirus vector.
The gene of human C1r has been expressed in a baculovirus-insect-cell system via the pAc373 transplacement vector. The full-length cDNA copy was inserted into the pAc373 vector downstream from the strong polyhedrin promoter of the baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells were cotransfected with the resultant plasmid, pAcC1r, and the wild-type AcNPV DNA. Recombinant viruses, which drove the expression of C1r protein, were selected by plaque morphology and ELISA. Insect cells infected with the recombinant virus produced and secreted human C1r protein, at a level of 1-2 mg/l of medium. The expressed C1r was isolated from the medium by chromatofocusing. On reducing gels only a single Coomassie-staining band was observed, and this band migrated at 80-83 kD characteristic of the unactivated C1r proenzyme. Its identification as C1r was immunologically confirmed on Western blots. C1 reconstituted from purified C1r expressed in insect cells together with human C1q and C1s proved biologically active in a hemolytic assay. Thus, the baculovirus-insect-cell system is capable of expressing and secreting a sophisticated, multifunctional human complement subcomponent in its biologically activatable form.
Gál
P
P
Biological Research Center, Hungarian Academy of Sciences, Budapest.
Sárvári
M
M
Szilágyi
K
K
Závodszky
P
P
Schumaker
V N
VN
D000818:Animals; D015139:Blotting, Southern; D015923:Complement C1r / Q000502:physiology*; D015941:Complement Hemolytic Activity Assay; D004792:Enzyme Precursors / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006461:Hemolysis / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007304:Insect Viruses; D010957:Plasmids; D014162:Transfection
D004792:Enzyme Precursors; D015923:Complement C1r