Studies on the propeptides of Streptomyces transglutaminases

Autor: Wen-Cheng Chang, 張文政
Rok vydání: 2013
Druh dokumentu: 學位論文 ; thesis
Popis: 101
Transglutaminase is an acyl-transferase enzyme, which catalyzes the formation of covalent bond between inter- or intra molecule of proteins. It has been widely used in biomedical and food processing. Previous studies in this laboratory have demonstrated that active form mature TGase can be obtained by co-expression of Trx-proTGase and tobacco vein mottling virus (TVMV) protease in E. coli at 17℃. During purification by Ni2+-NTA affinity column, the cleaved propeptide was found to be co-purified with the mature TGase. To study the interaction between the propeptide and mature TGase, these two proteins were purified individually and inhibition assay was conducted. Results showed that Streptomyces mobaraensis (Sm) and Streptomyces kentuckense (Sk or Sn) mature TGases interact with its Trx-propeptide by competitive type inhibition. The inhibition of Trx-Smpropeptide to its mature TGase was stronger than that of Sk TGase. Random mutagenesis was carried out on Sm propeptide by error-prone PCR to construct mutants which keep their chaperone functions and had less or no inhibitory activities to their mature TGases. A total of 34 mutants were obtained by TGase activity screening, and mature TGases from these mutants were expressed and purified by Ni2+-NTA column. Among these mutants, #302, #672, #C12, #D50, #434, #767, and #583, were characterized to have higher TGase activities or less binding abilities to their mature TGases. Furthermore, point mutations were generated on selected amino acids of Sm propeptide. Results showed that both of the inhibitory and chaperone functions of Sm propeptide are decreased in Y12F mutant and almost completely abolished in Y12A mutant. Moreover, the binding ability of Trx-propeptide to mature TGase was significantly decreased when Asp22 or Asn27 of the Sm propeptide was replaced by Val and Ala, respectively. The results indicated that Tyr12, Asp22 and Asn27 of the Sm propeptide play important roles in folding and inhibitory activity of Sm mature TGase.
Databáze: Networked Digital Library of Theses & Dissertations
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