1. Facilitated folding and subunit assembly in Escherichia coli and in vitro of nucleoside diphosphate kinase from extremely halophilic archaeon conferred by amino-terminal extension containing hexa-His-tag
Masao Tokunaga, Tsutomu Arakawa, Matsujiro Ishibashi FEBS Lett . 2004 Jul 16;570(1-3):87-92. doi: 10.1016/j.febslet.2004.05.082.
We have previously reported that nucleoside diphosphate kinase (HsNDK) from extremely halophilic archaeon Halobacterium salinarum was expressed in Escherichia coli as a soluble, but inactive form and required high salt concentrations for in vitro folding and activation. Here, we found that fusion of extra sequence containing hexa-His-tag at amino-terminus of HsNDK (His-HsNDK) facilitated folding and activation of HsNDK in E. coli. This is a first observation of active folding of halophilic enzyme from extremely halophilic archaeon in E. coli. The in vitro refolding rate of His-HsNDK after heat denaturation was greatly increased over the native HsNDK. Folded His-HsNDK isolated from E. coli formed a hexamer in both 0.2 M and 3.8 M NaCl at 30 degrees C, while the native HsNDK purified from H. salinarum dissociated to dimer in 0.2 M NaCl. The observed hexameric structure in 0.2 M NaCl indicates that amino-terminal extension also enhances dimer to hexamer assembly and stabilizes the structure in low salt. These results suggest that positive charges in fused amino-terminal extension are effective in suppressing the negative charge repulsion of halophilic enzyme and thus, facilitate folding and assembly of HsNDK.
2. Recombinant production and affinity purification of the FraC pore forming toxin using hexa-His tag and pET expression cassette
Mohammad Hassan Kheirandish, Mehdi Imani, Mahnaz Azadpour, Hossein Zarei Jaliani Iran J Basic Med Sci . 2017 Apr;20(4):380-385. doi: 10.22038/IJBMS.2017.8577.
Objectives:A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification.Materials and methods:After PCR amplification usingNcoIandHindIII-harboring primers, the gene fragment was cloned intopET-28a(+).Escherichia coli BL21was used for expression of constructed vector and toxin expression was verified by SDS-PAGE. For one-step purification Ni-NTA sepharose affinity chromatography was employed. For examination of purified toxin function, RBC hemolytic test was conducted.Results:The results showed that the FraC-coding gene was successfully cloned betweenNcoIandHindIIIrestriction sites and purified with affinity chromatography. Densitometric analysis represented the purity of approximately 97%. Hemolytic test indicated the purified FraC had remarkable lytic activity on RBC and almost lysed 50% of cells at the concentration value of 6.25 nM.Conclusion:The results indicated that not only purified toxin preserved its activity during expression and purification processes but also exerted its function at lower concentrations so that even the 0.09 nM displayed hemolytic effect.
3. Carboxymethylated polyethylenimine modified magnetic nanoparticles specifically for purification of His-tagged protein
Wangjie Lv, Xianzhe Shi, Mengmeng Chang, Qian Qin, Xiaoshan Sun, Bohong Wang, Guowang Xu, Tian Xia J Sep Sci . 2019 Feb;42(3):744-753. doi: 10.1002/jssc.201800969.
Employing immobilized metal-ion affinity chromatography and magnetic separation could ideally provide a useful analytical strategy for purifying His-tagged protein. In the current study, a facile route was designed to prepare CMPEI-Ni2+@SiO2@Fe3O4(CMPEI=carboxymethylated polyethyleneimine) magnetic nanoparticles composed of a strong magnetic core of Fe3O4and a Ni2+-immobilized carboxymethylated polyethyleneimine coated outside shell, which was formed by electrostatic interactions between polyanionic electrolyte of carboxymethylated polyethyleneimine and positively charged surface of 3-(trimethoxysilyl)propylamin modified SiO2@Fe3O4. The resulting CMPEI-Ni2+@SiO2@Fe3O4composite nanoparticles displayed well-uniform structure and high magnetic responsiveness. Hexa His-tagged peptides and purified His-tagged recombinant retinoid X receptor alpha were chosen as the model samples to evaluate the adsorption, capacity, and reusability of the composite nanoparticles. The results demonstrated the CMPEI-Ni2+@SiO2@Fe3O4nanoparticles possessed rapid adsorption, large capacity, and good recyclability. The obtained nanoparticles were further used to purify His-tagged protein in practical environment. It was found that the nanoparticles could selectively capture His-tagged recombinant retinoid X receptor protein from complex cell lysate. Owing to its easy synthesis, large binding capacity, and good reusability, the prepared CMPEI-Ni2+@SiO2@Fe3O4magnetic nanoparticles have great potential for application in biotechnological fields.
