Expression And Purification Of Docking Protein 5

Dok is the downstream protein of tyrosine kinase,which is important in cell signal transduction.The family contains 7 members from Dok1 to Dok7,which have similar structural characteristics:PH domain at N-terminal,central PTB domain and C-terminal region.The PH domain can help some proteins to locate on the plasma membrane,the central PTB domain mainly binds to phosphorylated tyrosine,and the C-terminal can bind to signal molecules to regulate downstream signaling pathways.Dok5 is a member of the Dok family.Studies have reported that Dok5 can bind the Ret receptor tyrosine kinase(Ret)receptor and promote neuronal differentiation through Glial cell line-derived Neurotrophic Factor(GDNF)signaling pathway.It is also the substrate of the tropomyosin-related kinase(Trk)receptor,which can bind to TrkB/C through the tyrosine phosphorylation of PTB domain and activate the MAPK signaling pathway to participate in the differentiation and apoptosis of nerve cells.And can be phosphorylated by insulin receptor,participate in insulin signaling pathway.These signaling pathways play important functions in the development of the nervous system,so it is speculated that Dok5 is a neurodevelopment-related protein.However,there have been no reports on the functional and molecular mechanism of Dok5 in the body.First,we analyzed the sequence of Dok5 gene and the amino acid sequence that it encodes.Dok5 is 921 bp in length and contains 8 exons and 7 introns.Encoding 306 amino acids,including PH domain,PTB domain and C-terminal region.Its relative molecular weight is 35.453 kDa,theoretical pI is 9.0,and its amino acid sequence is highly conserved in different species.SignalP signal peptide and TM transmembrane domain predictions showed that Dok5 had neither signal peptide structure nor transmembrane region.The online analysis of ProtParam and Protscale showed that the instability coefficient of Dok5 was 42.69 and the total average hydrophilic coefficient was-0.519.Although the C-terminal was rich in hydrophobic water,it was still a stable hydrophilic protein on the whole.According to NetPhos3.1 Server’s online prediction,Dok5 contains 50 potential phosphorylation sites,including 33 serine phosphorylation sites,14 threonine phosphorylation sites and 3 tyrosine phosphorylation sites.As a signaling protein,Dok5’s numerous phosphorylation sites are likely to act as docking sites for signaling molecules,activating downstream signaling pathways.The basic information provided guidance for subsequent protein expression.On the one hand,in order to find Dok5 interacting proteins and reveal their molecular mechanism in signal transduction process,we carried out the expression of proteins in different domains of Dok5 in the prokaryotic expression system.The cDNA encoding Dok5 full-length(FL)and PTB/PH/C domains were amplified by PCR,and cloned into expression vector pGEX 6P-1.The prokaryotic expression plasmids pGEX 6P-1-DOk5 FL,pGEX 6P-1-DOk5 PH,pGEX 6P-1-DOk5 PTB and pGEX 6P-1-DOk5 C were obtained.The recombinant plasmid was transformed into E.coli BL21,and strains expressing GST fusion protein were screened.The expressions of Dok5 FL,Dok5 PH and Dok5 PTB proteins were obtained from Escherichia coli BL21 at 0.1mM IPTG and 16℃,and purified by Glutathion Sepharose 4B affinity chromatography column.Western blot detection with Dok5 specific antibody showed that the purified fusion protein had good reactivity with Dok5 antibody,which laid an experimental foundation for the subsequent search for Dok5 interaction protein through GST pull down.On the other hand,prokaryotic expression proteins lack post-translational modification and cannot be correctly folded active proteins.In order to study Dok5 function at the in vivo level,it is essential to obtain antibodies that can specifically recognize Dok5.At present,the large-scale production of antibodies are polyclonal antibodies produced by immunizing animals with a c-terminal part of the synthetic peptide,which can recognize prokaryotic expressed proteins,but have no good specificity in mammalian cells and animal tissues.However,due to the post-translational modification function of insect expression system,the obtained protein is closer to the natural protein,and the antibody prepared from this antigen can specifically recognize the natural protein.Therefore,Dok5 FL and C-terminal proteins were expressed in the insect cell expression system for the subsequent preparation of antibodies as antigens.Dok5 FL and C terminal were cloned into pFastBac1 vector,respectively,and pfastBacl-Dok5 FL and PfastBac1-Dok5 C plasmid were constructed.The recombinant plasmid was transformed into DH10 competent cells,and the high-efficient bacmid-producing strain was obtained through blue-white spot screening.The insect cells SF21 were transfected with Bacmid to obtain P1/P2/P3 virus,respectively.High Five cells were infected with P3 virus in order to obtain a large number of intracellular expressed target proteins.Meanwhile,since Dok5 has no signal peptide structure,the signal peptide named hemolin was introduced into the pFastBacl vector,and PfastBacl-Hemlin-Dok5 FL and PfastBacl-Hemlin-Dok5 C plasmids were constructed to try to secret and express the protein,improve the efficiency of protein expression and purification,and thus provide active protein antigen for the preparation of Dok5 specific antibody.