Prokaryotic Expression,Purification And Activity Analysis Of Xentry-Cytoglobin Protein

It is has been proved that Cytoglobin has biological activity of antioxidant,antitumor and others,and may act as clinical potential medicine.But its molecular weight is far more than which can go through the cell membrane whose molecular weight requirements is less than 500 Da.More seriously for it has a tendency to form a dimer trimers and other polymers,etc.,which greatly limits its ability to enter cells.Our lab has previously constructed prokaryotic expression vector pET22b-TC and purified the targeted protein whose biological activity also studied i vitro and in vivo for further studies.TAT protein transduction domain amino acids sequence are YGRKKRRQRRR,is the shortest transduction effective sequence of TAT.Xentry whose amino acids sequence is LCLRPVG is a newly reported polypeptide with transmembrane ability,which is shorter than TAT.It has a good perforation effect.So we look forward to building fusion protein XC who can help more cytoglobin play its effect due to that more cytoglobin are in a state of inside cells.This study is significant in which prokaryotic expression vector pET22b-XC was successfully constructed.The recombinant XC protein was well expression by lactose induce.However,it was possible that isoelectric point of the targetd recombinant protein is too close to the isoelectric point of protein in escherichia coli itself.It is hard to isolate and purify the recombinant protein.Then for achieving more biologically active recombinant protein and for easier to isolate and purify,pET22b-pelB-XCHwas constructed who contains the pelB signal peptide and histidine tag.And so was pET22b-pelB-XLCH in which linker is added.But they were not significantly expressed by lactose and IPTG induction.Then try to use nickel column adsorption to collect targeted protein but did not get it.In order to obtain high expression levels Intracellular expression was tried.Then pET22b-XCH and pET22b-XLCH were successfully constructed.However the expression result is similar to before.Finally use pET28 a instead of pET22 b.PET28a-XCH and PET28a-XLCH were successfully constructed.It has show that recombinant protein was significantly expressed by IPTG induction with former vector.Then to obtain XC protein,Nickel column was used,by which collect protein in cell supernatant after ultrasound.Preliminary visible light analysis showed that the protein had biological activity.Then biological activity of XC had been explored in vitro.In summary,the result of this study demonstrated that significant ability of penetrating cell memberane may promote the intracellular function of cytoglobin.In vitro,XC has showed better biological activity than TAT-Cytoglobin for some studies but some are not.What are the differences between XC and TC whose mechanism into cells are explicit.All need futher studies.