title: Cloning, expression and purification of DNA-binding protein Mvo10b from Methanococcus voltae.
abstract: Mvo10b from the mesophilic archaeon Methanococcus voltae is a member of the Sac10b family which may play an important role in the organization and accessibility of genetic information in Archaea. Since Mvo10b is a DNA-binding protein as the other member in the Sac10b family, to obtain a recombinant Mvo10b requires an efficient and inexpensive expression and purification system for producing the protein free of nucleic acid contamination. Previously, the hyperthermophilic archaeal Ssh10b of the Sac10b family was successfully purified. However, the protocol adopted to purify Ssh10b is not appropriate for purifying the mesophilic Mvo10b. This study describes the successful expression and purification of the recombinant Mvo10b. The expression of recombinant Mvo10b was carried out in Escherichia coli, and the target protein was expressed in the soluble form. The protein was purified by polyethyleneimine (PEI) precipitation followed by nickel ion metal affinity chromatography. The purity of Mvo10b was checked to insure being free of nucleic acid contamination. The final protein yield is about 30mg/l of LB culture. The ensemble of NMR and far-UV CD data shows that the purified Mvo10b has abundant regular secondary structures and is correctly folded, which may have similar 3D structure as its hyperthermophilic counterpart [P62A]Ssh10b. The developed protocol has potential application in the production of the other thermophilic and mesophilic proteins in the Sac10b family.
authors.affiliation: Dept. of Biological Science and Technology, University of Science & Technology Beijing, 30 Xueyuan Road, Beijing 100083, China. jinsongxuan@gmail.com
authors.forename: Jinsong
authors.initials: J
authors.lastname: Xuan
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yao
authors.forename: Yingang
authors.initials: Y
authors.lastname: Feng
authors.forename: Jinfeng
authors.initials: J
authors.lastname: Wang
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D017017:Methanococcus / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004268:DNA-Binding Proteins