Expression and characterization of recombinant mouse β2-microglobulin type A in insect cells infected with recombinant baculoviruses

Abstract

The murine β 2-icroglobulin a cDNA was cloned into pAc373 and pVL941 transfer vectors and introduced via homologous recombination into the genome of Autographia californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. Both types of recombinant baculoviruses were isolated and used to infect Spodoptere frugiperde (Sf9) lepidopteran cells. β 2m was synthetized at a substantially higher in cells infected with the pVL941-derived virus than when the pAc373-based virus was used. β 2m was secreted into the culture medium where it accumulated and, under the best conditions, reached an approximate level of 10 μg/10 6 cells. Pulse-chase experiments after metabolic labelling with 35S-methionine followed by immunoprecipitation showed that β 2m was stable, but that the secretion processes in infected cells was relatively slow. Recombinant β 2m was endowed with biological activity and was indistinguishable from that produced by mouse cells in 2D gel analysis. β 2m was purified to near homogeneity from serum-free culture medium conditioned by recombinant baculovirus-infected cells by using an immunoaffinity column. The use of the insect cell/baculovirus expression system should constitute a suitable source of mouse β 2m and should aid experiments aimed at unravelling its interactions with mouse class 1 histocompatibility molecules.