Characterization of the single-stranded DNA binding protein pVVGJΦ of VGJΦ phage from Vibrio cholerae

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Abstract

pVVGJΦ, a single-stranded DNA binding protein of the vibriophage VGJΦ was subject to biochemical analysis. Here, we show that this protein has a general affinity for single-stranded DNA (ssDNA) as documented by Electrophoretic Mobility Shift Assay (EMSA). The apparent molecular weight of the monomer is about 12.7 kDa as measured by HPLC–SEC. Moreover, isoelectrofocusing showed an isoelectric point for pVVGJΦ of 6.82 pH units. Size exclusion chromatography in 150 mM NaCl, 50 mM sodium phosphate buffer, pH 7.0 revealed a major protein species of 27.0 kDa, suggesting homodimeric protein architecture. Furthermore, pVVGJΦ binds ssDNA at extreme temperatures and the complex was stable after extended incubation times. Upon frozen storage at − 20 °C for a year the protein retained its integrity, biological activity and oligomericity. On the other hand, bioinformatics analysis predicted that pVVGJΦ protein has a disordered C-terminal, which might be involved in its functional activity. All the aforementioned features make pVVGJΦ interesting for biotechnological applications.

Research highlights

► pVVGJΦ a DNA binding protein of phage VGJΦ has a general affinity for ssDNA. ► SEC revealed a major species of 27.0 kDa, suggesting a homodimeric protein. ► pVVGJΦ binds ssDNA at extreme temperatures, stable after extended times. ► After a year at –20 °C the protein kept its biological activity and oligomericity. ► pVVGJΦ is interesting and attractive for further application in biotechnology.

Introduction

The single-stranded DNA binding protein (SSB) of phage VGJΦ was previously identified to be encoded by ORF112 [1] and named pVVGJΦ [2]. This protein, isolated to homogeneity, binds ssDNA but not dsDNA [2]. However, a deeper biochemical characterization of this protein is necessary to rule out its biological function.

A previous mass spectrometry analysis showed a peptide sequence of a predicted molecular weight of 12.72 kDa, which corresponds to the predicted amino acid sequence of the open reading frame ORF112 VGJΦ [1], without the N-terminal methionine. Accordingly, pVVGJΦ was prominently expressed in V. cholerae infected with VGJΦ [1].

SSB proteins are characterized by the presence of a central conserved oligosaccharide/oligonucleotide binding (OB) fold comprising 5 antiparallel β-sheets that selectively bind ssDNA in a cooperative and non-sequence specific manner [3]. OB domains bind ssDNA in a cleft formed primarily by β-strands, by using aromatic residues that stack against nucleotide bases, and positively charged residues that form ionic interactions with DNA backbone [4]. The N- and C-terminal regions of the protein support effective interactions among functional entities of the SSB and other cell proteins involved in DNA metabolism, just to guarantee their important role in DNA replication, recombination and repair [5]. SSB proteins protect DNA from nucleases, stabilize ssDNA intermediates that are generated during DNA processing and prevent formation of DNA secondary structures [6]. Due to this array of essential functions SSB proteins are found in all living organisms and in viruses.

SSB proteins have found numerous applications in analytical molecular biology methods. They have been used to increase amplification efficiency with a number of diverse templates, prevent and reduce primer dimer formation, one of the problems known to cause inhibition of primer hybridization to the template and reduction of the number of primer molecules available for annealing [7]. They also interact efficiently with RNA, allowing a dramatic increase in the size of the cDNA synthesised by the reverse transcriptase activity of T. Termophilus DNA polymerase [8]. Provided the relevant biological role of SSBs and their broad spectrum of applications, a biochemical characterization of pVVGJΦ is a novel and valuable result. This paper shows the oligomeric state, the ssDNA-protein reaction kinetics and the effect of temperature on the DNA–protein interaction as well as stability of purified pVVGJΦ upon storage at −20 C for a year.

Section snippets

Isolation and purification of pVVGJΦ

pVVGJΦ was obtained from Vibrio cholera and purified under denaturing conditions as previously described [2]. SDS-PAGE (15% polyacrylamide) was used to monitor the process and the protein was quantified by Lowry. Native PAGE was run in a separating gel having a discontinuous polyacrylamide concentration gradient cast of 6 and 10%. The molecular size and degree of protein purity were estimated from densitometric scans of Coomassie brilliant blue stained gels, using a GENE GENIUS Gel

Molecular weight and isoelectric point determination of pVVGJΦ

The molecular weight of pVVGJΦ was determined by HPLC–SEC into a Superdex 200 HR 10/30 column. Only three peaks were observed. The major peak was eluted at 46.3 min (Fig. 1a) with a calculated molecular weight of 27 kDa. This value corresponds closely to the expected MW of the dimeric form of the predicted gene VVGJΦ product (25.44 kDa). Also, another minor peak was detected at 50.9 min with an estimated molecular weight of 6.66 kDa, which agrees with half of monomer molecular mass. The appearance

Discussion

The present study has shown that pVVGJΦ protein kept its homodimeric architecture after a year. The external protein-protein interactions predicted by ISIS that involve some residues in the N-terminal region could contribute to stabilize the dimeric structure. Similar results were reported in the gVp homodimer of the filamentous bacteriophage M 13 [3]. In that case, the structure of the dimer was primarily stabilized by hydrophobic interactions between residues in the N-terminal part, around

Conclusions

The single-stranded DNA binding protein pVVGJΦ of the vibriophage VGJΦ was characterized as a homodimeric protein having a disordered C-terminal. The protein binds ssDNA at extreme temperatures, even stable after extended incubation times. Frozen storage at − 20 °C after a year does not affect protein integrity, biological activity and oligomericity, so that further application in biotechnology i.e. for enhancing PCR assays is being considered.

Acknowledgments

We thank Kenny Batista for his assistance regarding the online submission the manuscript.

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