Expression And Purification Of Feline E3Protein Complex In Prokaryotic System

FIV attracts lots of scientists since it was found. There are a lot of comparabilitybetween FIV and HIV-1, from the genome of the virus, virus life cycle and the clini-cal symptom of infection. FIV can be used as an animal model to promote the HIV-1inhibitor research. The study shows that the FIV Vif performs the similar mechanismcompared with the HIV-1Vif, FIV Vif also combines with the Cullin5-ElonginB-ElonginC protein complex, forming the E3complex to mediate the degradation offeline A3protein. In this article, taking the FIV Vif crystal structure as the finalpurpose, I try to improve the solubility of FIV Vif by co-express the FIV Vif-ElonginB-ElonginC E3protein complex and looking forward to acquire the highpurity protein complex through prokaryotic system in vitro. So it will be a greatpromotion for the design of HIV-1inhibitors if the crystal structure of FIV Vif can beanalyzed.It’s hard to get a sufficient amount of the high purity FIV Vif protein, becausethe protein FIV Vif appears low solubility when it expresses alone using the pST39prokaryotic expression vector. In order to improve the solubility of FIV Vif, we co-express three exogenous gene, FVif, Elongin B, and Elongin C together so they canbond each other and forming the E3protein complex. Although the solubility of thisE3protein complex was improved obviously, the following purification result showslow purity, probably caused by the misfolding of FIV Vif. In order to acquire highpurity FIV Vif/EB/EC E3complex protein, we make an analysis and comparisonaiming at the protein sequence of FIV Vif and HIV-1Vif through Discovery studioand VectorNTI software. Finally11truncations aimed at FIV Vif were designed,FVifdN24, FVifdN58, FVifdN115, FVifdN149, FVifdN183, FVIFdN8/dC241FVIFdN8/dC226, FVIFdN30/dC241and FVIFdN60/dC241. The construction oftruncation dN58, dN30/dC226, dN60/dN226shows negative results, and dN8/dC226,N60/dC241protein complex didn’t make it induced by IPTG. The remaining seventruncations were purified by AKTA system after the detection of protein expression.Our results show that FVifdN24and FVifdN149have a good purification effect, thepurity of FVifdN24protein complex reach to80percent after gel filtration chromatography and FVif dN149makes it more than90. After large-scale purification,these two high purity protein complexes were sent to the cooperation unit for proteincrystallizing, but neither of the two protein complexes grows a satisfactory crystal,only FIV Vif dN149protein complex appears tiny crystals.CBF-β, as a necessary component of HIV-1Vif E3complex, was muchconcerned since it was found. In my research, in order to check whether feline CBF-βis an essential part for FVif E3complex, I verified the interaction of FIV Vif andCBF-β in prokaryotic system and eukaryotic system. In eukaryotic systems, theinteraction of FIV Vif and CBF-β was verified through immune-precipitate, co-transferring FIV Vif and CBF-β into CrfK cell. In prokaryotic system, FIV Vif, CBF-β, Elongin B and Elongin C were inserted into pST39co-expression vector, and thedirect interactions between FIV Vif and CBF-β were analyzed through identifying thecomponents of the Ni affinity elution sample. The results show that CBF-β can’tinteract with FVif-ElonginB-ElonginC to stabilize the protein complex. Probably,CBF-β is not essential in the degradation of FIV Vif E3complex-mediated felineAPOBEC3proteins.