Expression And Purification Of Hepatitis B Virus X Protein (HBx) In Escherichia Coli

BackgroundHepatitis B virus(HBV)infections cause chronic hepatitis and hepatocirrhosis,which are the major risk factors for hepatocellular carcinomas(HCC).Hepatitis B virus X protein(HBx)containing 154 amino acids is a highly conserved protein encoded by HBV genes.It is reported that HBx transcriptional activity plays an important role in HBV replication.Similarly,HBx also plays a role in the development of HCC by participating in multiple biological processes such as transcription,signal transduction,apoptosis,and protein degradation.It has been reported that the interactions between DDB1 and HBx contribute to viral infection and viral genome replication,gene instability and apoptosis.In 2010,Li,T.et al.solved a complex structure of full-length DDB1 and HBx alpha helix motif(88-100aa),which indicated that HBx binds to DDB1 through this highly conserved region and forms a new complex with Cul4-DDB1-Roc1 ubiquitin ligase.In 2016,Decorsiere,A.et al.found that HBx promotes the degradation of Smc5/6 protein complex,an inbitor of HBV gene replication by hijacking host Cul4-DDB1-Rocl ubiquitin ligase.Recent studies have shown that protein regions outside the HBx alpha helix motif also play an important role in maximizing HBV replication.Due to lacking the structural information on HBx full-length,the mechanisms on HBx full-length binds its interacting proteins and its role in promoting HBV replication remain unclear.In addition,HBx also interacts with other proteins,including HBxIP,Siah-1,and so on.The N-terminus of HBxIP can bind HBx on its 137-140aa region.In the midterm of cell division,HBxIP specifically forms a complex with the C-terminus of HBx,and both are located in the centrosome.Siah-1,an E3 ubiquitin ligase,is capable of promoting HBx polyubiquitination.Siah-1 degrades full-length HBx,but not HBx with C-terminal truncation,indicating HBx C-terminal truncation is more oncogenic.When the HBV genome is integrated into the host genome,it is often broken and truncated.In 2015,Wu,Q.et al.found overexpression of HBx truncations 1-127aa and 43-154aa in HBV-infected tumor tissues.These two truncations promote HBV replication and transcription.No matter expressed in insect cells or bacteria cells,HBx full length or truncations are all expressed in inclusion bodies,only a very small part of the protein is soluble.In the past,HBx protein was purified from inclusion bodies by denaturation-refolding method.However,the denaturation-refolding process may damage the tertiary structure of proteins,especially for cysteine-rich proteins such as HBx.denaturation-refolding HBx is not suitable for protein crystallization experiments.As mentioned in the literatures,maltose binding protein(MBP)is originated from Escherichia coli.Fusing this tag to the N-terminus of the target protein greatly improves protein expression level and assists protein properly folding.As an oligomer,MBP-HBx wild-type prevent protein from forming protein crystals,yet cannot bind its binding partners in vitro.ObjectiveIn order to solve the three-dimensional structure of the HBx recombinant protein,we optimized the condition for obtaining a large amount of soluble HBx recombinant protein(monomer)in Escherichia coli system,and perform crystallization experiments to obtain protein crystals.Methods1、Screening multiple tag proteins to find a proper one that can improve the soluble expression level of the target protein in Escherichia colil.2、According to the literatures,two stable HBx truncations 1-127aa and 43-154aa were constructed.MBP tag was added to the N-terminus of the target protein to obtain the recombinant protein expression plasmid.3、The hydrophobic interface of HBx was predicted by Swissmodel software,and the monomeric recombinant protein was obtained by point mutation experiment.4、The best expression plasmid was expressed in Escherichia coli.The recombinant protein was purified by affinity chromatography,ion exchange chromatography and size exclusion chromatography.5、High concentration and high purity HBx recombinant protein was used for protein crystallization experiment.The crystallization conditions of recombinant protein were screened by crystallization kit.Results1、As reported in the literatures,we found that Gst or His tag protein cannot increase the soluble protein expression level or improve the stability of the recombinant HBx protein in solution.However,fusing MBP tag protein can significantly improve the protein expression level and protein stability.2、After a series of experiments,we determined that His-MBP-HBx 43-154aa is the best expression plasmid.Since wild-type recombinant protein is oligomer,which denies crystallization.According to the prediction of Swissmodel software,a number of amino acids on the hydrophobic interface of the protein were mutated.Finally,we obtained the best combination as F73D/F132D/V133D.With this combination,we obtained the monomeric recombinant protein(34.48%).3、After a series of protein purification experiments,we obtained HBx recombinant protein with high concentration and high purity(10.27mg/ml,target protein ratio accounted for 72.10%).Through the screening of crystal reagent kits,we obtained spindle-shaped protein crystals.The crystallization reagents included pH 8.5(0.1 M,Bis-tris,propane),Sodium,Citrate(0.1M)and PEG 3350(30%).The crystal grows at the temperature of 20 ℃ in 14 days.Innovation Points1、After comparing several expression plasmids,we found that the best one that expresses His-MBP-HBx 43-154aa.The protein with high stability was suitable for the large-scale expression and purification of HBx.2、To our knowledge,our group was the first to obtain the monomeric HBx recombinant protein by point mutagenesis.Using this technique,we then obtain spindle-shaped protein crystals.