Expression Of Adenylate Kinase Fused Mouse Ubiquitin Active Enzyme In Escherichia Coli And Its Application In Ubiquitination

Ubiquitination is a post-translational modification of proteins that is widely present in eukaryotes.It is a multi-enzyme cascade reaction involving E1 ubiquitin-activating enzyme,E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase enzyme.And other molecules are also involved in this process,such as ATP and Mg²⁺.The ubiquitination process is related to various physiological functions,such as cell proliferation and differentiation,signal transduction,protein localization,antiviral,DNA damage repair,autophagy,growth and development,and apoptosis.In human cells,there are 2 E1ubiquitin-activating enzymes,about 40 E2 ubiquitin-conjugating enzymes,and approximately 600 E3ubiquitin ligase enzymes.In vitro ubiquitination reaction is very important in the study of ubiquitination modification.It is difficult to solve how to obtain a large number of active E1 to meet the requirements of in vitro ubiquitination.In vitro ubiquitination reactions,UBA1 is usually used as E1 to perform the ubiquitination reaction,so a large number of active UBA1 needs to be obtained to meet the requirements of experiments and the preparation of ubiquitin products.At present,there are two methods to purify UBA1.The first way is purified by UBA1 richly in tissue.The second is purified by overexpression in insect cells or E.coli.But it only obtains a little UBA1 though those two ways.We improved the expression of UBA1?mUBA1?in E.coli by protein fusion expression.Meanwhile,the fused mUBA1 was more active than the non-fused mUBA1 enzyme in some ubiquitination reactions.Adenosine kinase?adk?is a protein found in almost all organisms from bacteria to mammals.It can catalyze 2 moles of ADP to produce 1 mole of ATP and 1 mole of AMP.Adk was highly expressed in E.coli.The expression of the target protein can also be significantly increased by fusing adk with other proteins.The expression level of the fusion protein adk-mUBA1 was significantly higher than that of the proteins expressed in other literatures by fusing adk with mUBA1 and the expression level was about 10 times of mUBA1.The protein we obtained also increased a lot by increasing the expression of mUBA1.mUBA1 obtained was about 1-3 mg of pure protein in 1 L mUBA1 solution,while adk-mUBA1 obtained about 15-20 mg.By contrast,the amount of protein obtained increased by about 8 times.Adk-mUBA1 and mUBA1 were obtained by optimizing the conditions of affinity purification and anion exchange.Adk-mUBA1 and mUBA1 have same ubiquitin-activiting activity and there is no significant difference in either UBA1 binding to Ub through sulfide ester bond,UBA1 transferring ubiquitin activity to UbcH5B,Ube2S directly forming ubiquitin chain activity,or UbcH5B transferring ubiquitin to different types of E3 forming ubiquitin chain activity.The activity of adk-mUBA1 is obviously stronger than that of mUBA1 in some reactions.Adk-mUBA1 can have both adenylate kinase activity and mUBA1 ubiquitin-activating activity after adk is fused,so it can hydrolyze ADP by adk to provide ATP to mUBA1 to activate Ub and initiate ubiquitination reaction.A large number of ubiquitination reactions involve cell lysates,but the cell lysate contains a large amount of ATP hydrolase.Exogenously supplied ATP will be hydrolyzed to ADP in a very short time,thus unable to supply mUBA1 to activate Ub.There are two strategies to solve the problem:1.Cell membrane components in cell lysate were removed?S100?.A large amount of ATP hydrolase exists in the cell membrane,and the removal of cell membrane components will greatly reduce the ATP hydrolase in the lysate,but a lot of membrane proteins will be lost,and the effect is not very well at the same time.ATP was regenerated through ADP through the addition of inositol kinase and phosphoinositol in the ATP regeneration system.The experiment found that this strategy only worked in S100,and the effect was not significant in the whole cell lysate.Adk-mUBA1 was able to synthesize twice as many ubiquitin chains as mUBA1 by ubiquitination in cell lysates by contrast.It was found that adk-mUBA1 could also generate UbcH-O-Ub twice as much as mUBA1.Adk-mUBA1 can activate Ub by ADP,so it is more suitable for long-term ubiquitination than mUBA1.Adk-mUBA1 can be used in a large number of experiments related to the synthesis of E2-O-Ub complex by catalytic synthesis of E2-O-Ub complex.E2-O-Ub with a purity of about 90%was obtained for experimental study by tandem affinity purification.Adk-mUBA1 can also be used in chemical synthesis,such as in vitro synthesis of ubiquitin chain,to meet the needs of various experiments.