Silkworm Small Heat Shock Protein 19.9 And Its N-terminal Deletion Mutants Of The Prokaryotic Expression And Molecular Chaperone Function

The small heat shock proteins (sHsps) are stress-inducible molecular chaperones that range in size from 12 to 43 kDa. Although the sHsps family vary greatly in sequence and size, but there are several common structure features: 1,α-crystallin domain composed of approximately 90 amino acid residues; 2, the content of Cys lower than other protein family; 3, forming large oligomers with a dynamic four structure; 4, the molecular chaperone activity of inhibiting protein aggregation.Our experiment selected sHsp19.9 which has been found as the smallest sHsp in Bombyx mori. By bioinformatics analysis, we constructed three pET-28a expression vectors respectively inserted with full-length and two truncation mutants of deleting 1-24 and 1-62 N-terminal amino acids residues. They were transformed into E.coli BL21-Star to express inclusion bodies which were purified with metal affinity chromatography and refolded with Sephedex G-150 gel column. Polyclonal antibody was generated by immuning a New Zealand male rabbit with recombinant sHsp19.9-FL protein and the titer was over 1:12 800, measured by ELISA. According to SDS-PAGE (reduced and non-reduced) and Western blot, sHsp19.9-FL, sHsp19.9-△1 and sHsp19.9-△2 all formed dimers through intramolecular disulfide bonds. Accordingly, the experiment analyzed that they were all degraded by trypsin sensitivity experiment, indicating that none of the three proteins formed stable structure of large oligomers. While the molecular chaperone activity by citrate synthase (CS) thermal aggregation experiments and the DTT-induced lysozyme aggregation, we confirmed that the obtained sHsp19.9-FL protein possessed the molecular chaperone function, but others didn't. The experiment results give more information about the relationship between the structure and function of small heat shock proteins from the silkworm.Through analysis of Western blot and semi-quantitative ELISA, the stress expression of the spinning silkworm and pupa were different, which were incubated at room temperature for different period after heat treatment. However, the expression level after heat treatment was higher than before. The total RNA and protein in several tissues of the fifth instar silkworm were extracted, and the expression level of sHsp19.9 was analyzed using real time PCR and Western blot. The results showed that the tissues in rich expression were the strong ones in growth and development process, such as the testis. Its amount was much higher than other tissues, which indicated that sHsp19.9 may be involved in cell division, differentiation and other activities of life. The stress expression of sHsp19.9 in silkworm and specific tissues provided the basis for further researches on the mechanism of its function.