Prokaryotic Expression, Purification Of The SIR-2.1Protein And Screening Its Specific Affinity Ligand By The Phage Peptide Library Display

The Sirtuin family has ADP-nucleic acid transferase activity and HDACactivity. The HDAC reaction is to substrate acetyltransferase toADP-ribosylation of NAD+. Along with the reaction，one molecule of NAD+splits into a molecule nicotinamide (NAM) and one molecule ofO-acetyl-ADP-ribose. Sirtuin mediates two catalytic reaction：the fracture anddeacety of NAD+. The ADP-ribose transferase activity of Sirtuin is to transferacetylated proteins to ADP-ribose of NAD+.Sirtuin ADP-mediated nucleic acidtransfer, and deacetylation, may be initially deacetylation reaction to produceADP-ribose, and it is added in the subsequent process of the HDAC into the endmaterial. In addition, maybe some of the Sirtuin members only haveADP-ribose transferase activity.Yeast SIR2is firstly discovered in Sirtuin family members.Many earlierstudies had made people aware of SIR2could combine with other proteins toform complex，allow it to fulfil its function of gene silencing,but the discoveryof acylation enzyme clarifies its mechanism.SIR2plays a key role in restraining rDNA recombination, telomerase gene silencing in yeast,and mating-type genesilencing in the rDNA silencing.The relationship between SIR2and longevity isanother important reason for its attention.SIR2activator or adding a copy of thesir2gene can delay the aging of yeast so as to prolong the life of yeast.Recent studies have confirmed, calorie restriction can also improve thenematode life largely,and SIR-2.1as expression of homologous protein of SIR2protein family in nematode also has a large degree of upregulation inquantity.Research shows that limited extension of SIR-2.1nematode life withheat may be an important life related proteins.But recent studies have shownthat, SIR-2.1protein of Caenorhabditis elegans stress ability, aging andapoptosis plays an important role, is a very important life related protein, so itattracts more attention.Because the content of SIR2.1is less in the body, and is not convenient forthe detection of activity and for other aspects, so there is no prokaryoticexpression of related literature on SIR2.1now.The purpose of this paper is thesir-2.1gene was cloned into the expression vector of the nematode, transformedinto E.coli prokaryotic expression.Expression and protein purification of soluble with a histidine tag using nickel column, a molecular sieve to remove salt,purified soluble SIR-2.1protein.Then take it as target protein, using peptidelibrary affinity screening techniques,and screening the peptides which canspecific bind with it,through the amplification of plaque,extraction ofsingle-stranded DNA sequencing,get specific affinity peptide sequence,provideimportant information for peptide design and SIR-2.1specific affinity agonist,so the study of some anti-aging drugs and diseases associated with aging (suchas cancer, diabetes and some tissue degenerative disease) became possible drug.In this paper, through the construction of expression vector for SIR-2.1protein,it makes the protein expression of gene cloning become29aa-490aa oftotal protein which is on the expression vector,including the all the active site,lay the foundation for later screening.The induction of host bacteria SIR-2.1protein expression in IPTG conditions, the SIR-2.1protein was induced byadjusting the amounts of soluble expression, purification of expressed proteinswere identified by Western Blotting, to get a lot of soluble SIR-2.1proteinwith high purity.By phage display random peptide library screening, screeningfor random12peptide library to prokaryotic expression of SIR-2.1protein, twelve peptide sequences were screened out， and most of the sequences arepresented good regularity, and there are many valuable homologous motif, thesehomologous motif could specifically bind to SIR-2.1，and it provide importantstructural information for designing Short peptide which is binding with SIR-2.1protein specific agonists.