Differential expression of the Ebola virus GP(1,2) protein and its fragments in E. coli.
Bacterial expression platforms are frequently used for the expression and production of different recombinant proteins. The full length Ebola virus (EBOV) GP(1,2) gene and subfragments of the GP(1) gene were cloned in a bacterial expression vector as a C-terminal His(6) fusion protein. Surprisingly, the full length EBOV GP(1,2) gene could not be expressed in Escherichia coli. The subfragments of GP(1) were only expressed in small amounts with the exception of one small fragment (subfragment D) which was expressed at very high levels as inclusion bodies. This was seen even in the in vitro translation system with no expression of full length GP(1,2), GP(1) subfragments A and C and low level expression of subfragment B. Only the subfragment D showed high level of expression. In E. coli (Top10), the recombinant GP(1) subfragment D protein was expressed exclusively as an insoluble approximately 25 kDa His(6) fusion protein, which is the expected size for a non-glycosylated recombinant protein. The IMAC purified and refolded non-glycosylated protein was used to immunize mice for the development of monoclonal anti-EBOV antibodies which successfully yielded several monoclonal antibodies with different specificities. The monoclonal and polyclonal antiserum derived from the animals immunized with this recombinant GP(1) subfragment D protein was found to specifically recognize the full length glycosylated EBOV GP(1,2) protein expressed in mammalian 293T cells, thus, demonstrating the immunogenicity of the recombinant subfragment.
Das
Dipankar
D
University of Alberta, Edmonton, Alta., Canada T6G 2N8.
Jacobs
Fred
F
Feldmann
Heinz
H
Jones
Steven M
SM
Suresh
Mavanur R
MR
D000818:Animals; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006825:Hybridomas; D007114:Immunization; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C113189:envelope glycoprotein, Ebola virus