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

Abstract

Abstract. 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

“…Our findings coincide with data of Alba homologues from other hyperthermophilic Archaea [4], [7], [8], [17], [18]. Previously, the formation of the Alba1/Alba2 heterodimer was shown by NiNTA agarose chromatography in which only one of two Aeropyrum pernix Alba proteins was His-tagged, but both proteins co-eluted [11], as was reported for the Alba proteins from Sulfolobus solfataricus [8].…”

“…Our ITC experiments indicate that Alba1 binds DNA, but we could not experimentally measure binding of Alba2 using this method. The gel shift [11] in addition to the ITC experiments, shows that Alba2 condenses DNA. Other than the UV melting curves that show some preferences of Alba2 for AT-DNA and GC-DNA, our data do not generally confirm that Alba2 is DNA-sequence specific.…”

“…E. coli was cultivated at 46°C after activation of synthesis with 1 mM isopropyl-beta-D-thiogalactopyranoside. The proteins were purified as described previously [11], by combination of (NH 4 ) 2 SO 4 precipitation, NiNTA, ultrafiltration, and temperature-induced precipitation of impurities. Protein concentrations were determined with the Bio-Rad (Biorad, USA) and bicinchoninic acid (BCA; Pierce, USA) protein assays, with the BCA protein assay showing more accurate protein concentrations.…”

“…Agarose gel mobility shift assays (EMSAs) were used to examine the effects of binding of Alba1 and Alba2 with or without the His-tag to linear DNA molecules [11], [12]. A 2686 bp linearised DNA (pUC19/ Eco RI) was used.…”

Knockdown of ghAlba_4 and ghAlba_5 Proteins in Cotton Inhibits Root Growth and Increases Sensitivity to Drought and Salt Stresses

“…The Alba proteins have a distinctive property in the regulation and organization of the organism’s genomes through acetylation and deacetylation (Goyal et al, 2012). The Alba protein binding has a very high affinity towards double-strand deoxyribonucleic acid (DNA), thus named as acetylation lower binding affinity ( Alba ) (Crnigoj et al, 2011). In Sulfolobus solfatataricus , a species of thermophilic archaeon, Alba proteins have been found to reversibly acetylated at lysine 6 ( Lys16 ) by a homologous protein acetyltransferase (Pat) and deacetylated by a sirtuin family deacetylase ( Sir2 ) (Starai and Escalante-Semerena, 2004).…”