Purification and Characterization of a Novel Cold-regulated Protein from an Ice-nucleating Bacterium, Pseudomonas fluorescens KUIN-1

Abstract

The psychrotrophic ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1 respond to a decrease in temperature with the induction of proteins that are classified as cold shock proteins (CSPs). We found the function of a 26-kDa protein of the CSPs in the strain KUIN-1. In strain KUIN-1, a cold shock from 18 to 4°C induced the synthesis of the 26-kDa protein. By analysis with SDS-PAGE, it was then demonstrated that the 26-kDa protein was produced by the cells after treatment at 4°C. The 26-kDa protein was purified to apparent homogeneity by (NH₄)₂SO₄ precipitation and some chromatographies (QA52, phenyl Superose, Superose 12, and Mono Q). The purified 26-kDa protein is composed of 6 subunits of 26.5-kDa with a molecular mass of approximately 159-kDa according to gel filtration and SDS-PAGE. The N-terminal sequence of the 26-kDa protein was Gln-Ala-Ala-Tyr-Tyr-Pro-Ala-His-His-His-Gln-Gln-Val-Gln-Gln-His-Trp-Gly-His-His-. Specifically, 26-kDa protein of the CSPs of strain KUIN-1 was very effective in protecting the cold-labile enzyme, lactate dehydrogenase against denaturation by freezing. The characteristics of 26-kDa protein are analogous to the cold-regulated protein of the plants.

• ice-nucleating bacterium
• cold-regulated protein
• cold shock protein
• lactate dehydrogenase
• Pseudomonas fluorescens KUIN-1