Expression and Secretion of Active Recombinant Streptomyces Transglutaminase in Corynebacterium glutamicum

• Main Authors: Guan-Chi Wang, 王冠棋
• Other Authors: 楊明德
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/05205820624124547059

碩士 === 國立中興大學 === 分子生物學研究所 === 100 === Transglutaminase (protein-glutamine:amine γ- glutamyltransferase, EC 2.3.2.13; TGase) catalyzes irreversible translational modification of proteins, resulting in the formation of inter- or intramolecular cross-linkage. The aim of this study was to improve the yield of extracellular production of active Streptomyces TGase in Corynebacterium glutamicum. A typical signal peptide of the C. glutamicum Sec and Tat pathways, s949 and s1834, respectively, was chosen as secretory signal for Streptomyces TGase. However, replacement of the original CspA signal peptide with either s949 or s1834 exhibits no improvement in the yield of extracellular recombinant TGase in C. glutamicum. Moreover, the ability of pro-peptide to function as a molecular chaperone and act in trans to mediate efficient secretion of mature TGase from C. glutamicum was explored. Results showed that active mature TGase could hardly be detected in the culture medium when mature TGase and its pro-peptide were transcribed as a polycistronic mRNA. A metalloprotease (MPI) from S. netropsis with pro-TGase activation activity was previously identified and the mpI gene was cloned from the constructed genomic library. Co-expression of the cloned S. netropsis metalloprotease and pro-TGase in C. glutamicum revealed that a limited amount of processed mature TGase could be identified in the culture medium. To exclude the possibility that MPI is not the enzyme responsible for catalyzing pro-TGase activation in S. netropsis, a second metalloprotease (MP II) from S. netropsis was cloned and sequenced in this study. DNA sequence analysis revealed that S. netropsis mpII contains 1,575 bp and a polypeptide of 524 amino acids could be encoded with an estimated molecular weight of 55.2 kDa. The deduced amino acid sequence contains a typical HEXXH motif of metallopeptidase family. Introduction of S. netropsis mpII and co-expression with pro-TGase in C. glutamicum to improve the processing and secretion efficiency of pro-TGase is now under investigation. Furthermore, to enhance the extracellular production of S. netropsis TGase in E. coli, the first seven amino acids of S. netropsis pro-peptide was replaced with those from S. hygroscopicus TGase pro-peptide. By lowering the incubation temperature to 17℃ after IPTG induction, significant amounts of S. netropsis pro-TGase could be identified in the culture medium. This result clearly demonstrated that pro-peptide of S. netropsis has influence on the solubility and secretion of TGase.