Expression and characterization of recombinant mouse beta 2-microglobulin type a in insect cells infected with recombinant baculoviruses.
The murine beta 2-microglobulina cDNA was cloned into pAc373 and pVL941 transfer vectors and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus downstream of the polyhedrin promoter. Both types of recombinant baculoviruses were isolated and used to infect Spodoptera frugiperda (Sf9) lepidopteran cells. beta 2m was synthesized at a substantially higher rate in cells infected with the pVL941-derived virus than when the pAc373-based virus was used. beta 2m was secreted into the culture medium where it accumulated and, under the best conditions, reached an approximate level of 10 micrograms/10(6) cells. Pulse-chase experiments after metabolic labelling with 35S-methionine followed by immunoprecipitation showed that beta 2m was stable, but that the secretion process in infected cells was relatively slow. Recombinant beta 2m was endowed with biological activity and was indistinguishable from that produced by mouse cells in 2D gel analysis. beta 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 beta 2m and should aid experiments aimed at unraveling its interactions with mouse class I histocompatibility molecules.
Godeau
F
F
Unité de Biologie Moléculaire du Gène, INSERM U 277, Institut Pasteur, Paris.
Casanova
J L
JL
Fairchild
K D
KD
Saucier
C
C
Delarbre
C
C
Gachelin
G
G
Kourilsky
P
P
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D051379:Mice; D009036:Moths; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D001613:beta 2-Microglobulin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D012333:RNA, Messenger; D011994:Recombinant Proteins; D001613:beta 2-Microglobulin