A comparative study of different vector designs for the mammalian expression of recombinant IgG antibodies.
Monoclonal antibodies (Mab) are the fastest growing group of biopharmaceuticals in development. For production in mammalian cells, the four polypeptide chains of the immunoglobulin diheterotetramer must be assembled prior to exit from the endoplasmic reticulum. Various recombinant Mab expression vectors have been developed utilizing mono-and bicistronic expression cassettes encoded on one or two plasmids. However, there are only few studies providing information on the type of vector design optimal for stable or transient production of recombinant IgG. Consequently, in this study, we have constructed a series of mammalian expression vectors for the production of recombinant human or chimeric IgG antibodies with different expression cassette designs. Versions for monocistronic and bicistronic expression with different promoters and cistron arrangements were generated. Antibody production levels were evaluated in transiently transfected 293T and CHO-K1 cells. Furthermore, stable CHO cell lines were generated and analyzed for antibody production levels and stability. Our results indicate that compared to monocistronic expression, EMCV IRES-mediated bicistronic expression constructs yield similar antibody expression levels and show long-term stability in CHO cell lines. Addition of a third cistron encoding YFP was shown to facilitate screening and isolation of clones using a FACS sorter.
Li
Jiandong
J
Institut für Biochemie und Biotechnologie, Abteilung Biotechnologie, Technische Universität Braunschweig, Spielmannstr.7, 38106 Braunschweig, Germany.
Menzel
Christian
C
Meier
Doris
D
Zhang
Congcong
C
Dübel
Stefan
S
Jostock
Thomas
T
D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003587:Cytomegalovirus / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008970:Molecular Weight; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014162:Transfection
D020121:5' Untranslated Regions; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D008164:Luminescent Proteins; D020648:Peptide Elongation Factor 1; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria