here is my problem: i inserted the linear diubiquitin cDNA(which has two successive copy of the natural ubiquitin gene) into the PET22b plasmid between Ndel and XhoI site for obtaining the linear diubiquitin(which means the protein molecular weight is twice of the monoubiquitin).unfortuntly, i only got the mono ubiquitin, is there any tips on expression of such kind protein which has several successive copys of the same gene?
Multiple copies can be expressed from the same DNA. Are the two copies of you gene in frame with each other. Did you join them using a flexible linker like Gly-Ser-Gly-Ser? It could be that your linker is too short.
I looked at human ubiquitin PDB 1UBQ. ( see http://www.rcsb.org/pdb/explore.do?structureId=1UBQ ) You should ligate last basic Arg to linker Gly-Ser-Gly-Ser-Gly-Ser-Gly then to first residue of next ubiquitin (Met?). That should give enough length for the 2 proteins to fold.
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