Purification and characterization of a novel cold-regulated protein from an ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1

The psychrotrophic ice-nucleating bacterium, Pseudomonas fluorescens KUIN-1 respond to a decrease in temperature with the induction of proteins that a re 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 shoc k from 18 to 4 degrees C induced the synthesis of the 26-kDa protein. By an alysis with SDS-PAGE, it was then demonstrated that the 26-kDa protein was produced by the cells after treatment at 4 degrees C. The 26-kDa protein wa s purified to apparent homogeneity by (NH4)(2)SO4 precipitation and some ch romatographies (QA52, phenyl Superose, Superose 12, and Mono Q). The purifi ed 26-kDa protein is composed of 6 subunits of 26.5-kDa with a molecular ma ss 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-Hi s-His-His-Gln-Val-Gln-Gln-His-Trp-Gly-His-His-. Specifically, 26-kDa protei n of the CSPs of strain KUIN-1 was very effective in protecting the cold-la bile enzyme, lactate dehydrogenase against denaturation by freezing. The ch aracteristics of 26-kDa protein are analogous to the cold-regulated protein of the plants.