Send Orders for Reprints to reprints@benthamscience.net 266 Current Biotechnology, 2014, 3, 266-272 Expression, Purification and In Silico Studies of Cold Resistant Protein from Plant Growth Promoting Ps fluorescens Mutant CRPF1 Mahejibin Khan * and Reeta Goel Department of Microbiology, CBSH, GB pant university of Agriculture and Technology, Pantnagar, India Abstract: The production of cold shock proteins constitutes an adaptive response in psychrotrophic and psychrophillic microorganisms to allow survival after cold shock. In the present study a small 10kDa cold resistant protein (CRP) was expressed and purified by immuno-affinty chromatography from CRPF1, a cold resistant mutant of Pseudomonas fluorescens. Sequence analysis and homology studies of cold resistant gene revealed 91% sequence homology with the Pseudomonas fluorescens Pf-5 gene encoding cold shock DNA-binding protein. Three dimensional computational model of CRP was found similar to the known structures of cold shock proteins. It was consisted of five-stranded β- barrel-fold and highly conserved ribonucleoprotein consensus sequences, RNP1 (KGFGFI) and RNP2 (VHF) motif on its surface. Functional characterization of purified protein confirmed the DNA binding property of protein. From the sequence analysis and functional characterization of protein, it was concluded that a low molecular weight, DNA -binding cold resistant protein is induced in CRPF1 that helps in the survival of mutant under low temperature stress conditions. Keywords: Cold adaptation, cold resistant protein, plant growth promoting rhizobacteria. INTRODUCTION Hostile environments, such as extreme temperature (low and high), drought, salinity etc are detrimental to the plant development and thus affect the plant growth and the productivity of various crops around the world. Low temperature is the major factor, limiting the productivity and geographical distribution of many species, including important agricultural crops [1]. Therefore to deal with the adverse environmental factors, strategies such as increase in membrane fluidity, synthesis and accumulation of cryoprotectant and altered growth have been reported as protective measures in various plants. Moreover, use of plant growth promoting bacteria such as epiphytic or endophytic plant growth- promoting rhizobacteria (PGPR) has also been reported to protect plants from frost injuries. These bacteria enhance plant growth while improving their resistance to stress [2, 3]. Lindow & Leveau [4] reported the use of a combination of an antagonistic and an ice nucleation bacterium to manage biotic and abiotic stress in plants. A number of PGPR strains associated with the plant rhizosphere from genera Azospirillum, Alcaligens, Bacillus, Erwinia, flavobacterium [5], Rhizobium leguminosarum [6] and Pseudomonas [7] have been reported to exert beneficial effects on plants at low temperature by enhancing their growth and resistance to stress. These bacteria colonize in rhizosphere at temperatures at which only small numbers of microorganisms are able to proliferate and promote plant growth. The potential use of fluorescent pseudomonads at low temperature in plant growth promotion is well documented [7]. Berrios studied the effect of Antarctic strain *Address correspondence to this author at the CSIR-Central Food Technological Research Institute, Resource Centre Lucknow, India; Tel: 91 522 2818126; Fax: 91 522 2818126; E-mail: mahejibin@cftri.res.in of Pseudomonas sp. on Deschampsia Antarctica, a hairy grass that grows in Antarctic continent and found that the strain was able to solubilize different sources of phosphates and help in plant growth promotion through plant root development [8]. To overcome the deleterious effect of low temperature and for survival under adverse environmental conditions, cold adapted organisms have developed various molecular and physiological adjustments [9]. The outstanding common feature of the microbial response to low temperature in all the microorganisms including thermophiles [10], mesophiles [11,12], psychrotrophs [13] and psychrophiles [14] is the over expression of a specific subset of proteins: the cold- shock proteins (CSPs). These proteins are involved in various cellular functions such as transcription, translation, and regulation of protein folding. CSPs minimize secondary folding and act as DNA or RNA chaperons [10], thereby stimulating production of other cold inducible proteins [15,16]. In general CSPs are involved in establishing a new cell balance, which in turn facilitates survival at low temperature [14]. Mishra and Goel [17] developed cold resistant mutant of Ps. fluorescens and test their stress tolerance and plant growth promoting activities in various crops at 10˚C and 4˚C. These studies showed that these mutants could survive and stimulate plant growth even at 4˚C. On the basis of the above facts, this study was planned for the identification and functional characterization of CRP from cold resistant mutant of fluorescent pseudomonad CRPF1. We have cloned, purified and characterized a cold resistant protein from CRPF1. Amino acid sequence analysis and three dimensional structure reveal that CRP belongs to cold shock protein family and help in the survival of mutant strain under low temperature stress condition through its DNA binding property. 2211-551X/14 $58.00+.00 © 2014 Bentham Science Publishers