Molecular Cloning, Isolation, and Properties of Chaperone Skp from Yersinia pseudotuberculosis



E. V. Sidorin1, N. M. Tishchenko1, V. A. Khomenko1, M. P. Isaeva1, P. S. Dmitrenok1, N. Yu. Kim1, G. N. Likhatskaya1, and T. F. Solov’eva1


  1. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences


The skp gene of Yersinia pseudotuberculosis was expressed without its signal sequence in Escherichia coli BL21(DE3) cells. The recombinant protein Skp accumulated in soluble form in the cytoplasm of the producer strain. The protein was isolated and characterized: the molecular weight, isoelectric point, N-terminal amino acid sequence (20 amino acid residues), and the content of the secondary structure elements were determined. Using cross-linking stabilization and high-mass MALDI-TOF mass spectrometric analysis, it was found that rSkp forms a stable homotrimer in solution and interacts with human IgG. Three-dimensional models of the Skp trimer and its complexes with Fc- and Fab-fragments of human IgG1 were constructed by computer modeling.

CovalX Technology Used (Click each option to learn more)



Skp of Yersinia pseudotuberculosis was cloned, isolated and evaluated. To prepare for analysis by mass spectrometry, a matrix was obtained containing sinapic acid (10 mg/ml) in acetonitrile-0.1% TFA. Samples were mixed with the matrix and then spotted onto a target using the dried droplet method. The target was analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.



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