Isolation of Two Strong Poly (U) Binding Proteins from Moderate Halophile Halomonas eurihalina and Their Identification as Cold Shock Proteins

Cold shock proteins (Csp) are known to be expressed in response to sudden decrease in temperature. They are thought to be involved in a number of cellular processes viz., RNA chaperone activity, translation, transcription, nucleoid condensation. During our studies on ribosomal protein S1 in moderate halophile Halomonas eurihalina, we observed the presence of two strong poly (U) binding proteins in abundance in cell extracts from cells grown under normal growth conditions. The proteins can be isolated in a single step using Poly (U) cellulose chromatography. The proteins were identified as major cold shock proteins belonging to Csp A family by MALDI-TOF and bioinformatic analysis. Csp 12 kDa was found in both exponential and stationary phases whereas Csp 8 kDa is found only in exponential phase

Poly acrylamide gel electrophoretic analysis of poly (U) cellulose chromatography peak fractions.

<p>A. SDS-PAGE of fractions from exponential phase S-100. B. SDS-PAGE of fractions from stationary phase S-100. In both cases, Lane M: Molecular weight marker, lane 1: exponential/stationary phase S-100 (10 µl), lane 2: fraction number 3 (100 µl) and lanes 3–4: fractions 24 and 25 (100 µl each). C. Native-PAGE analysis. Lane 1–2: Fractions 24 and 25 of exponential phase S-100 elution, lane 3: Fraction 24 of stationary phase S-100 elution.</p

Identification of <i>H. eurihalina</i> poly (U) binding proteins by MALDI-TOF MS analysis.

<p>Identification of <i>H. eurihalina</i> poly (U) binding proteins by MALDI-TOF MS analysis.</p

Elution profile of S-100 on poly (U)-cellulose column.

<p>Fractions 1–20 were eluted with buffer A. Fractions 21–30 eluted with buffer B. Absorbance at 280 nm for the fractions eluted with buffer A shows the release of non-specific proteins bound to the poly (U) column. Single, sharp peak represents the fractions containing eluted poly (U) binding proteins.</p

Two-dimensional gel electrophoresis of poly (U) binding proteins isolated from S-100.

<p>A. Exponential phase. B. Stationary phase. Electrophoresis was performed as described under Methods.</p