Characterization of human DNA ligase I expressed in a baculovirus-insect cell system.
The baculovirus expression system was used to overexpress human DNA ligase I (hLig I). Approx. 2 mg of recombinant hLig I was produced per 10(8) Spodoptera frugiperda Sf9 insect cells infected with the recombinant baculovirus. The optimum time point for the production of biologically active recombinant hLig I was 48 h post-infection. Lig I activity was demonstrated by auto-adenylating, polynucleotide joining and DNA relaxation assays. The baculovirus system has the advantage over previously described methods for producing hLig I of generating large amounts of a full-length active protein.
Gallina
A
A
Istituto di Genetica Biochimica ed Evoluzionistica, CNR, Pavia, Italy.
Rossi
F
F
Milanesi
G
G
Rossi
R
R
Montecucco
A
A
Ciarrocchi
G
G
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002474:Cell-Free System; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007106:Immune Sera; D015151:Immunoblotting; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera
D007106:Immune Sera; C000607892:LIG1 protein, human; D011994:Recombinant Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP