Prolonged and increased expression of soluble Fc receptors, IgG and a TCR-Ig fusion protein by transiently transfected adherent 293E cells.
In studies of the relation between structure and function of proteins of the immune system, there is a continuous need for screening of a large number of protein variants. To optimise the yield following transient gene expression in small or medium culture volumes, several parameters were investigated. First, secretion levels of a soluble form of human Fcgamma receptor IIA (FcgammaRIIA) were measured after transfection of 293, 293E, 293T as well as COS-7 cell lines. The transgene was under cytomegalovirus (CMV) promoter control on the expression vector pcDNA3, which also contains an SV40 origin of replication (SV40 ori). All 293 cell lines secreted more protein than COS-7 cells. Introduction of the Epstein Barr virus (EBV) origin of replication (oriP) greatly increased the protein expression from the 293E cells, both the amount of protein produced per day and the duration of production. At best, 293E cells secreted fully functional protein for 3-4 weeks provided supernatant was harvested every 2-3 days followed by medium replacement. This method was then used for expression of soluble forms of human FcgammaRI, FcgammaRIIB, the human neonatal Fc receptor (FcRn), a T cell receptor (TCR)-immunoglobulin (Ig) fusion protein, and human IgG3. With an initial culture volume of 5 ml, the yield was approximately 200 microg for FcgammaRIIA, 1.5 microg for FcgammaRI, 5 microg for FcRn, 20 microg for FcgammaRIIB, 40 microg for the TCR-Ig fusion protein and 850 microg for IgG3. Culture expansion during the 3 weeks of culture further increased the yield. Protein yield was also improved by scaling up the initial volume. This approach can provide sufficient amounts of protein for screening experiments, and in the case of antibody, milligrams of recombinant protein for extensive structural analysis can be obtained from one single transient transfection. The approach should be of interest to laboratories that do not have access to a bioreactor but still have a requirement for reasonable amounts of protein to be produced in an easy and cost-effective manner.
Berntzen
Gøril
G
Department of Molecular Biosciences, University of Oslo, P.O.Box 1041, 0316, Oslo, Norway. goril.berntzen@bio.uio.no
Lunde
Elin
E
Flobakk
Morten
M
Andersen
Jan Terje
JT
Lauvrak
Vigdis
V
Sandlie
Inger
I
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D019556:COS Cells; D018929:Cell Culture Techniques / Q000379:methods; D002522:Chlorocebus aethiops; D003587:Cytomegalovirus / Q000235:genetics; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis*; D011401:Promoter Regions, Genetic; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis*; D011961:Receptors, Fc / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014162:Transfection; D019076:Transgenes
D000952:Antigens, Polyomavirus Transforming; D019309:Epstein-Barr Virus Nuclear Antigens; D007074:Immunoglobulin G; D011948:Receptors, Antigen, T-Cell; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; C417029:EBV-encoded nuclear antigen 1