ABSTRACT
Genome duplication in E. coli is carried out by DNA polymerase III, an enzyme complex consisting of ten subunits. Investigations of the biochemical and structural properties of DNA polymerase III require the expression and purification of subunits including α, ge, θ, γ, δ', δ, and β separately followed by in vitro reconstitution of the pol III core and clamp loader. Here we propose a new method for expressing and purifying DNA polymerase III components by utilizing a protein co-expression strategy. Our results show that the subunits of the pol III core and those of the clamp loader can be coexpressed and purified based on inherent interactions between the subunits. The resulting pol III core, clamp loader and sliding clamp can be reconstituted effectively to perform DNA polymerization. Our strategy considerably simplifies the expression and purification of DNA polymerase III and provides a feasible and convenient method for exploring other multi-subunit systems.
Subject(s)
Cloning, Molecular , DNA Polymerase III , Chemistry , Genetics , Metabolism , DNA Replication , DNA, Bacterial , Genetics , Escherichia coli , Genetics , Plasmids , Metabolism , Polymerization , Protein Engineering , Methods , Protein Subunits , Chemistry , Genetics , Metabolism , Recombinant Proteins , Chemistry , Genetics , MetabolismABSTRACT
E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.
ABSTRACT
E.coli single-stranded DNA-binding protein (SSB) plays an important role in replication, recombination and repair of DNA and is thus crucial for the survival of the bacteria.We described a high expression and efficient purification scheme and kinetic assay of interaction with its substrate, single-stranded DNA (ssDNA). A ssb gene (537 bp) for encoding SSB was obtained by PCR amplification from E.coli K-12 genome. The expression vector of the fusion protein SSB was constructed by attaching ssb gene to pQE30. SSB fusion protein was expressed in M15 E.coli strain induced by IPTG. SDS-PAGE analysis revealed that the expected protein with a molecular weight 20.6kDa was soluble and amounted to about 30% of the total bacterial protein. SSB protein was purified by immobilized metal (Ni2+) chelation affinity chromatography and the purity was about 90%. The resulting SSB protein was a correctly folded tetramer analyzed by gel filtration. It could bind ssDNA with equilibrium dissociation constant (KD) of 4.79×10-7 mol/L as determined by surface plasmon resonance.
ABSTRACT
Fusion tags are originally developed to facilitate the purification of recombinant protein from crude extracts. In recent years, the discovery of different tags and the development of fusion strategy make the function of fusion tags diversified. However, there was no a cure-all fusion tag for different applications. We here give an overview of fusion tag technology and the different applications of fusion tags, including the purification, detection and oriented immobilization of recombinant protein, the visualization of bioevent in vivo, the enhancement of the yield of protein, the improvement of the solubility and stability of the expressed protein.
Subject(s)
Recombinant Proteins , Chemistry , SolubilityABSTRACT
DNA mismatch repair gene mutS (2.56 kb) was PCR modified and cloned into a secretive prokaryotic expression vector pET32a (+) which carries a N-terminal His.tag + and thioredoxin sequence. MutS protein was expressed with high level after IPTG induction using the strain E. coli AD494(DE3). SDS-PAGE revealed that the expected protein with a molecular weight of 108 kD which is about 35% of the total bacterial proteins is almost soluble. The expected protein was purified directly by immobilized metal (Ni2+) chelation affinity chromatography and the purity is over 90%. MutS protein activity verified using mismatch DNA showed that the expression product can recognize and bind to base-pair mismatch specifically.