Overexpression, purification and characterization of RecJ protein from Thermus thermophilus HB8 and its core domain

Abstract

A recJ homolog was cloned from the extremely thermophilic bacterium Thermus themophilus HB8. It encodes a 527 amino acid protein that has 33% identity to Escherichia coli RecJ protein and includes the characteristic motifs conserved among RecJ homologs. Although T.thermophilus RecJ protein (ttRecJ) was expressed as an inclusion body, it was purified in soluble form through denaturation with urea and subsequent refolding steps. Limited proteolysis showed that ttRecJ has a protease-resistant core domain, which includes all the conserved motifs. We constructed a truncated ttRecJ gene that corresponds to the core domain (cd-ttRecJ). cd-ttRecJ was overexpressed in soluble form and purified. ttRecJ and cd-ttRecJ were stable up to 60 degrees C. Size exclusion chromatography indicated that ttRecJ exists in several oligomeric states, whereas cd-ttRecJ is monomeric in solution. Both proteins have 5'-->3' exonuclease activity, which was enhanced by increasing the temperature to 50 degrees C. Mg(2+), Mn(2+) or Co(2+) ions were required to activate both proteins, whereas Ca(2+) and Zn(2+) had no effects.

Figure 1

Amino acid sequence of the T.thermophilus, E.coli and Aquifex aeolicus RecJ proteins. PPX1 of S.cerevisiae and the PRUNE gene product of D. melanogaster are also aligned according to Aravind and Koonin (8). Identical amino acid residues are indicated by white letters on black. The conserved aspartate and hisitidine residues of five motifs are indicated at the top by asterisks. The cd-ttRecJ region (40–463) is indicated at the top by two arrows. ScPPX1, PPX1 of S.cerevisiae; dmPRUNE, PRUNE gene product of D.melanogaster. Motifs I–V indicate the five conserved motifs described in Aravind and Koonin (8).

Figure 2

Purification of ttRecJ and cd-ttRecJ and limited proteolysis of ttRecJ. (A) Purification of ttRecJ. Lane 1, total cell extract; lane 2, supernatant of the total cell extract; lane 3, extract from the pellet; lane 4, Ni²⁺–nitriloacetate–agarose; lane 5, refolding fraction; lane 6, MonoQ HR5/5. (B) Limited proteolysis of ttRecJ with thermolysin. An aliquot of 10 µM ttRecJ was reacted with 0.5 µg/ml thermolysin in 20 mM Tris–HCl buffer, pH 8.0, containing 2 mM CaCl₂. This reaction mixture was incubated at 25°C for the indicated incubation periods. Lane 1, ttRecJ; lanes 2–4, digests incubated for the periods indicated above each lane. The arrow indicates the core domain of ttRecJ. (C) Purification of cd-ttRecJ. Lane 1, supernatant of the total cell extract; lane 2, Ni^2+–nitriloacetate–agarose; lane 3, Bio-Gel HTP gel; lane 4, MonoQ HR5/5; lane 5, HiPrep 16/60 Sephacryl S-200 HR. Each step was analyzed by staining the SDS–PAGE gel (12% w/v acrylamide) with Coomassie brilliant blue. The molecular mass markers were: MBP–paramyosin, 83 kDa; glutamic acid dehydrogenase, 62 kDa; aldolase, 47.5 kDa; triosephosphate isomerase, 32.5 kDa; β-lactoglobulin A, 25 kDa; lysozyme, 16.5 kDa.

Figure 3

CD spectra and thermostabilities of ttRecJ and cd-ttRecJ. (A) Far-UV CD spectra. Measurements were performed at 25°C in buffer solution at pH 7.2 containing 1.6 µM ttRecJ or 1.5 µM cd-ttRecJ, 50 mM potassium phosphate, 100 mM KCl, 0.1 mM DTE and 0.1 mM EDTA. (B) Temperature dependence of the molar ellipticity at 222 nm ([θ]₂₂₂). Measurements were perfomed in the same solutions used for the far-UV CD spectra measurements. The heating rate was 1°C/min. Thick line, ttRecJ; thin line, cd-ttRecJ.

Figure 4

Size exclusion chromatography of ttRecJ (A) and cd-ttRecJ (B). ttRecJ and cd-ttRecJ were loaded onto a Superdex 200 HR 10/30 column equilibrated with 20 mM Tris–HCl, 100 mM NaCl, 0.1 mM EDTA, 10 mM 2-mercaptoethanol and 10% glycerol, pH 7.5. ttRecJ and cd-ttRecJ were analyzed by absorbance at 280 nm with a flow rate of 0.5 ml/min. The molecular size markers (Sigma) are: potato β-amylase, 200 kDa; yeast alcohol dehydrogenase, 150 kDa; bovine serum albumin, 66 kDa; bovine carbonic anhydrase, 29 kDa; horse heart cytochrome c, 12.4 kDa.

Figure 5

Exonuclease activity of ttRecJ and cd-ttRecJ. (A) An aliquot of 50 nM 5′-³²P-labeled ssDNA was reacted with 0.1 µM ttRecJ at 37°C. The reaction mixture contained 20 mM Tris–HCl, 10 mM MgCl₂, 100 mM KCl and 1 mM DTT, pH 7.5. The degradation of ssDNA was analyzed by denaturing PAGE (15% polyacrylamide). Lane 1, ssDNA; lanes 2–6, ssDNA reacted with ttRecJ for the incubation period indicated above each lane. The 49nt arrow indicates the substrate 49mer ssDNA and the 1 nt arrow the released 5′-end nucleotide. (B) An aliquot of 20 nM 5′-³²P-labeled ssDNA was reacted with 0.1 µM ttRecJ and cd-ttRecJ at 25°C for 30 min. The reaction mixture contained 20 mM Tris–HCl and 100 mM KCl, pH 7.5, with the addition of 10 mM MgC1₂ or MnCl₂. Lanes 1 and 5, ssDNA; lane 2, with ttRecJ; lane 3, with ttRecJ in the presence of MgC1₂; lane 4, with ttRecJ in the presence of MnCl₂; lane 6, with cd-ttRecJ; lane 7, with cd-ttRecJ in the presence of MgC1₂; lane 8, with cd-ttRecJ in the presence of MnCl₂. (C) Time course of ttRecJ exonuclease activity. The reaction mixture was as in (A). The percentage of undegraded DNA was plotted against incubation time. Open circle, 37°C; closed circle, 50°C.

Figure 6

Kinetic analysis of the exonuclease activities of ttRecJ (A) and cd-ttRecJ (B). An aliquot of 10 nM 5′-³²P-labeled ssDNA was reacted with ttRecJ and 1 nM 5′-³²P-labeled ssDNA with cd-ttRecJ. The reaction mixture containing 20 mM Tris–HCl, 10 mM MgCl₂, 100 mM NaCl and 1 mM DTT, pH 7.5, was incubated at 25, 37 and 50°C. The rates of ssDNA degradation by both proteins were plotted against each concentration of protein. Closed square, 25°C; closed circle, 37°C; closed triangle, 50°C. Solid lines show the theoretical curves (see Materials and Methods).