Expression Of Anti-DR5SCFV And Multivalent Antibody In Pichia Pastoris

IntroductionTumor necrosis factor-related apoptosis inducing ligand(TRAIL) is a member of the Tumor necrosisfactor (TNF) superfamily, and it has been one of the main research goals in cancer treatment in recent years.TRAIL has5kinds receptors in the surface of the cell, Death Receptor4(DR4), Death Receptor5(DR5),“decoy” receptor1(DcR1),“decoy” receptor2(DcR2) and osteoprotegerin(OPG). Fas-associated proteinwith death domain (FADD) motif is found on the protein of DR4and DR5, and DISC formates throughFADD combining with procaspase-8and-10. DISC induces the cleavage and activation of FADD, then itcan activates protein to caspase, induces apoptosis of cancer cells. We established monoclonal antibodyagainst DR5, i.e.YM366EC. We have amplified the variable regions of VL and VH of anti-DR5antibodyusing PCR technology, and generate scFv and tetramer, which was designated3D and3S, respectively. Wehave proved that the multivalent antibody, i.e.3S, has anti-tumor effect and proliferation-inhibiting activity.The scFv gene fragment was linked with gene of P53to obtain fused gene, P53can be automaticallyaggregated into the tetramer, so it can formate tetravalent. And in this experiment, we obtained divalentantibody, they was aggregated via disulfide bonds and α-helix structure, formate forward tetravalentantibody and reverse tetravalent antibody, which designated3ZS and3FS, respectively. In addition, wechoose the Pichia pastoris expression system instead of Eukaryotic expression system and Prokaryotic cellexpression system to express the antibody protein. At present, most proteins which expressed by Pichiapastoris are methanol-inducible useing methanol as a carbon source in the fermentation process. Sincemethanol is toxic, volatile, flammable, there is some risk in the procedure. So the constitutive expressionwas used to select pGAPZαC as an expression vector. GAP is its promoter and can promote the expressionof target genen through useing glucose as a carbon source in this expression system. Compared witheukaryotic expression system, the biggest advantage is low-cost, high antibody production, short cultivationperiod. Compared with prokaryotic cell expression system, it has a better protein translation modification.ObjectiveUse Pichia pastoris to express scFv and multivalent antibody of YM366EC. MethodsUsing PCR technology, the gene fragment of3D and3S was amplified from plasmid ofpSegtag2A-3D and pSegtag2A-3S respectively and inserted into the Pichia yeast expression vector ofpGAPZαC. Then using Overlap extension PCR technology, the gene sequence of forward tetravalent andreverse tetravalent antibody sequence were amplified, VL-VH-HSA-VL-VH-IgG hinge-dhlx andVL-VH-HSA-VH-VL-IgG hinge–dhlx, designated3ZS and3FS, respectively. Subcloned them into Pichiayeast expression vector pGAPZαC by EcoR Ⅰa nd NotⅠ restriction sites, therefore pGAPZαC-3D,pGAPZαC-3S, pGAPZαC-3ZS, pGAPZαC-3FS were produced. The authenticity of the fused gene wasconfirmed by sequencing, then Pichia pastoris GS115was transformed to express fused protein, positiveclones were screened with bleomycin (zeocin). Using broth PCR technology and Western blot to verifywhether the target gene is successfully transferred and the target protein s is successfully expressed or not,then expand the cultivation of positive clone, purify expressed proteins by Ni2+-NTA super flow affinitycolumn from the supernatant of culture. Using PCR technology, the human DR5gene fragment wasamplified from pcDNA3.1-DR5. Then subcloned it into prokaryotic cell expression vector PET-30a byBamH I and Hind III restriction sites, and therefore PET-30a-DR5were produced. The authenticity of thefused gene was confirmed by sequencing, then E.coli BL21was transformed to express fused protein, using0.1mmol/L IPTG induced the expression of DR5and purifying proteins DR5by Ni2+-NTA super flowaffinity column. Using DR5package ELISA plate, detect the affinity of the antibody.ResultsFragments of scFv, tetramer, forward tetravalent and reverse tetravalent anti-DR5antibody wereamplified, pGAPZαC-3D, pGAPZαC-3S, pGAPZαC-3ZS, pGAPZαC-3FS were produced. The fusedprotein have expressed in Pichia pastoris GS115. Western blot showed that p-3D, p-3S, p-3ZS and p-3FSexpressed protein with molecular weight41.3KD,46.9KD,2.4KD and72.4KD. Though ELISA assay,3Shas highest affinity, followed3ZS and3FS, the lowest of the affinity is3D.ConclusionCompared with single-chain antibody, anti-DR5multivalent antibodies has higher affinity:3S hashighest affinity, followed3ZS and3FS, the lowest of the affinity is3D.