Cloning of P<inf>II</inf> -like gene and purification of P<inf>II</inf> protein from nitrogen-fixing Paenibacillus Polymyxa

Abstract

The P<inf>II</inf> proteins are signal transduction elements that integrate the signals of nitrogen, carbon and energy and transducer this information to proteins involved in nitrogen metabolism. P<inf>II</inf> a trimeric protein encoded by the gln B gene, is a component of the adenylation cascade involved in the regulation of glutamine synthetase (GS). P<inf>II</inf> is a protein of about 110 amino acid residues extremely well conserved. In this work a 250 pb P<inf>II</inf> encoded gene fragment was PCR amplified from two Paenibacillus polymyxa (P. polymyxa) strains (M and G) using universal degenerate primers. Cloning, sequencing and nucleotide analysis revealed that the protein fragment of our two strains have encoded an 87 residues of amino acids. 77 a. a(s) residues are identical with a. a of P<inf>II</inf> protein from P. polymyxa in gene bank giving 80% similarity (accession No. AY773941). M and G strains showed four new a. a(s) in B loop and new 6 a. a(s) in T loop. The P<inf>II</inf> protein was eluted via colum after extracted form bacterial cells expressed P<inf>II</inf> gene in high concentration 200, 250 mM Using ion exchange chromatography and sodium chloride gradient 0 - 300 mM. The purified protein were analysed on 15 % SDS-PAGE and gave sharp band at ~ 14 kDa and that was confirmed by western blot analysis with specific antibodies raised against P<inf>II</inf> protein. © 2013 by Nova Science Publishers, Inc. All rights reserved.