Heterologous Expression of the Alba Protein from the Hyperthermophilic Archaeon Aeropyrum Pernix

Nucleic acid binding proteins have important roles in DNA and RNA packaging, stabilisation and repair, and in gene regulation, and they are therefore essential for all organisms. All of the known hyperthermophiles have at least one DNA sequence encoding for the Alba proteins. The Alba proteins are small (approximately 10 kDa), DNA-binding, basic proteins that appear to partly compensate for the lack of histones in the archaea Aeropyrum pernix and other hyperthermophiles. Two sequences of these potential histone counterparts, the Alba proteins, were identified in the Aeropyrum pernix genome (APE1832.1 and APE1823). By using a wide range of experimental techniques and by examining several combinations of expression systems the expression of recombinant Alba1 and Alba2 proteins was optimized. Co-expression of both of the Alba proteins was needed when isolating recombinant Alba2. The purification of both recombinant Alba1 and Alba2 His-tagged proteins were simplyfied in satisfactory yield. The electrophoretic mobility shift assay demonstrated the ability of the Alba1 and Alba2 proteins from Aeropyrum pernix to bind DNA. (doi: 10.5562/cca1772

Heterologous Expression of the Alba Protein from the Hyperthermophilic Archaeon Aeropyrum Pernix

Nucleic acid binding proteins have important roles in DNA and RNA packaging, stabilisation and repair, and in gene regulation, and they are therefore essential for all organisms. All of the known hyperthermophiles have at least one DNA sequence encoding for the Alba proteins. The Alba proteins are small (approximately 10 kDa), DNA-binding, basic proteins that appear to partly compensate for the lack of histones in the archaea Aeropyrum pernix and other hyperthermophiles. Two sequences of these potential histone counterparts, the Alba proteins, were identified in the Aeropyrum pernix genome (APE1832.1 and APE1823). By using a wide range of experimental techniques and by examining several combinations of expression systems the expression of recombinant Alba1 and Alba2 proteins was optimized. Co-expression of both of the Alba proteins was needed when isolating recombinant Alba2. The purification of both recombinant Alba1 and Alba2 His-tagged proteins were simplyfied in satisfactory yield. The electrophoretic mobility shift assay demonstrated the ability of the Alba1 and Alba2 proteins from Aeropyrum pernix to bind DNA. (doi: 10.5562/cca1772

Interactions of archaeal chromatin proteins Alba1 and Alba2 with nucleic acids.

BACKGROUND: Architectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823). METHODOLOGY/PRINCIPAL FINDINGS: THESE TWO PEPTIDES WERE EXPRESSED AND THEIR INTERACTIONS WITH VARIOUS DNAS WERE STUDIED USING A COMBINATION OF VARIOUS EXPERIMENTAL TECHNIQUES: surface plasmon resonance, UV spectrophotometry, circular dichroism-spectropolarimetry, gel-shift assays, and isothermal titration calorimetry. CONCLUSIONS/SIGNIFICANCE: Our data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs

UV melting curves of GC-DNA.

<p>The molar ratios of protein:DNA base pairs were at 1∶30 to 1∶2 for Alba1 (a), at 1∶30 to 1∶10 for Alba2 (b), and at 1∶30 to 1∶20 for the Alba1/Alba2 complex (c).</p

Native and SDS PAGE electrophoresis.

<p>Left: Native protein PAGE electrophoresis with 20% homogeneous gel for the Phast system. á, equinatoxin II; b´, Alba1; c´, Alba2; and d´, equimolar ratio of Alba1/Alba2. Right: SDS PAGE electrophoresis shows influence of disulphide bridges (±DTT) on dimerisation of Alba proteins. a, h, PageRuler protein ladder; b, e, Alba1; c, f, Alba2; d, g, equimolar ratio of Alba1/Alba2.</p

Sequence composition of the Alba proteins.

<p>The amino-acid sequences of Alba1 (APE1832.1–APA1) and Alba2 (APE1823–APA2) from <i>A. pernix</i> was aligned with those of the homologous proteins from <i>Sulfolobus shibitae</i> Alba1 (P60849– SSA1) and <i>S. solfataricus</i> Alba2 (Q97ZF4–SSA2). The positively charged amino acids (R and K), phenylalanine (F), tyrosine (Y) and cysteine (C) are marked.</p

CD spectra of Alba1/Alba2 complex.

<p>Molar ellipticity, [Θ], in the far-UV range (200–250 nm) at different temperatures (as indicated), for the Alba1/Alba2 complex without bound DNA (a), with CT-DNA (b), with AT-DNA (c), and with GC-DNA (d). Molar ratio of Alba:DNA per base pair was 1∶5 at pH 7.0 (50 mM NaH₂PO₄).</p

CD spectra of Alba1.

<p>Molar ellipticity, [Θ], in the far-UV range (200–250 nm) at different temperatures (as indicated), for Alba1 without bound DNA (a), with CT-DNA (b), with AT-DNA (c), and with GC-DNA (d). The molar ratio of Alba:DNA per base pair was 1∶5 at pH 7.0 (50 mM NaH₂PO₄).</p

UV melting curves of AT-DNA.

<p>The molar ratios of protein:DNA base pairs were at 1∶30 to 1∶5 for Alba1 (a) and at 1∶30 to 1∶2 for Alba2 (b) and the Alba1/Alba2 complex (c).</p