Engineering the assembly pathway of the baculovirus-insect cell expression system.
The synthesis of complex biological structures such as antibodies using recombinant DNA technology is a major biotechnological advance. Active murine antibody (IgG) oligomers, composed of two heavy (H) and two light (L) polypeptide chains, have been expressed and secreted by the baculovirus-insect cell expression system. Unfortunately, expression of the functional antibodies is accompanied by the formation of abnormal protein complexes and aggregates in which the polypeptide chains are bound together into incorrect associations. The formation of these abnormal complexes or protein aggregates in insect cells may be caused by insufficient intracellular levels of two catalytic proteins, immunoglobulin binding protein (BiP or GRP78), and protein disulfide isomerase (PDI). Consequently, we obtained the genes coding for murine BiP and PDI and cloned the genes into the baculovirus vector (Autographa californica nuclear polyhedrosis virus) to obtain AcBB-BiP and AcBB-PDI. Infection of Spodoptera frugiperda (Sf-9) insect cells with these two baculoviruses yielded recombinant proteins of the correct size that were recognized by antibodies to these proteins. Cloning these genes into the baculovirus vector is one approach to engineering the assembly pathway in order to lower aggregation and increase production of functionally active proteins and oligomers.
Hsu
T A
TA
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218.
Eiden
J J
JJ
Betenbaugh
M J
MJ
D000818:Animals; D000917:Antibody Formation / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D000091342:Endoplasmic Reticulum Chaperone BiP; D015870:Gene Expression; D005818:Genetic Engineering; D005822:Genetic Vectors; D006360:Heat-Shock Proteins; D007535:Isomerases / Q000096:biosynthesis / Q000235:genetics; D018832:Molecular Chaperones; D009036:Moths; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D002352:Carrier Proteins; D004274:DNA, Recombinant; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases