Analysis of IgG heavy chain to light chain ratio with mutant Encephalomyocarditis virus internal ribosome entry site.
Immunoglobulin G (IgG) is a heterotetrameric protein assembled from two identical heavy chain (HC) and two identical light chain (LC) polypeptides. The HC and LC folding and assembly are a crucial step for IgG production. It is affected by the ratio of HC to LC expression (HC:LC). To date, the HC:LC ratio was analysed mainly by cotransfection of different amounts of two monocistronic HC and LC expression plasmids, an approach biased by different transfection efficiencies. To circumvent this problem, a series of Encephalomyocarditis virus internal ribosome entry site (EMCV IRES) variants with different translation efficiencies were created and used to mediate HC translation in bicistronic constructs. HC and LC were translated from the same mRNA, which provides a more accurate method for the evaluation of the optimal ratio of HC:LC. The results show that the IgG optimal expression levels were obtained when the IRES mediated translation efficiency of the HC was about 50% compared to the cap-dependent translation of the LC. A surprisingly sharp transition to low production was shown when the ratios were below 40%. This study provides a new method to investigate the production of heterodimeric proteins in mammalian cells and adds understanding to the mechanisms of IgG folding and assembly.
Li
Jiandong
J
Institut für Biochemie und Biotechnologie, Abteilung Biotechnologie, Technische Universität Braunschweig, Spielmannstr. 7, 38106 Braunschweig, Germany.
Zhang
Congcong
C
Jostock
Thomas
T
Dübel
Stefan
S
D001483:Base Sequence; D002460:Cell Line; D004686:Encephalomyocarditis virus / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005453:Fluorescence; D015870:Gene Expression; D017930:Genes, Reporter; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation; D014176:Protein Biosynthesis; D017510:Protein Folding; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D008164:Luminescent Proteins