An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies.
Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein-protein, and DNA-protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione-agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor.
Aatsinki
Jyrki T
JT
Institute of Dentistry, University of Oulu, P.O. Box 5281, FIN-90014, Oulu, Finland. Jyrki.Aatsinki@oulu.fi
Rajaniemi
Hannu J
HJ
D000906:Antibodies / Q000235:genetics* / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics; D011974:Receptors, LH / Q000276:immunology; D011993:Recombinant Fusion Proteins; D012995:Solubility
D000906:Antibodies; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase