Studies on the heterologous proteins expressed in bacillus subtilis

• Main Author: 陳美樺
• Other Authors: 葉娟美
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/49698391676282699607

碩士 === 國立中興大學 === 食品科學系 === 90 === Abstract (part I) Bacillus subtilis can secrete the enzymes into the supernatant in large amounts and has been used in the production of industrial enzymes for a long time. It is also as excellent host for the secretory production of heterologous proteins. Coexpression of two structure genes in one host is necessary in some cases. One is to get a functional recombinant protein which is consisted of two subunits. Others such as the protein of interest must be coproduced with accessory factors such as molecular chaperones and transcription activation factors to enhanced the yield of active heterologous proteins. There are mainly two strategies to coexpress heterologous proteins. One is to use a bicistronic vector, which is generated by cloning two target genes into one plasmid. The other strategy is to use two plasmids in which foreign genes are cloned in two different vectors. In this study, we use two plasmid coexpression system to ease the control of heterologous protein production. Plasmids used in this study included pME, pHG and pEX5B. The plasmid pME came from the shuttle vector pMK4 with part of subtilisin YaB gene, ale E inserted into it. pEX5B and pHG came from the shuttle vector pHY300PLK, with subtilisin YaB gene and CGTase gene inserted into it separately. pHG or pEX5B was transformed into Bacillus subtilis DB104 with pME. Our goal is to study the possibility of coexpression with pHY300PLK-based vector and pMK4-based vector in Bacillus subtilis. The results suggested that pHY300PLK-based vector and pMK4-based vector can coexpress heterologous proteins in Bacillus subtilis DB104. The segregation and structure stability are high. Based on this coexpression system, other coexpression system can be developed in the future. Abstract ( part II ) Bacillus subtilis can secrete proteins directly into the growth medium, this ability makes Bacillus subtilis an attractive host for expression of heterologous recombinant proteins. Although Bacillus subtilis has this advantage over other host such as Escherichia coli, its secretion of heterologous proteins is not so efficient in many cases. In this study, we aimed to study the effects of histidine-tag to the expression and secretion of heterologous proteins in Bacillus subtilis. In our prior studies, the signal peptide of Subtilisin YaB was fused to the N terminus of the type I antifreeze protein. Although the signal peptide can lead Subtilisin YaB to cell membrane for processing during secretion in Bacillus subtilis. The expression level of antifreeze protein was difficult to observe. Then, we replaced the original promoter with a strong σA type promoter. We found that the expression of the antifreeze protein elevated but located in the cell membrane and insoluble fraction. In this study, we questioned the effect of histidine-tag in the C-terminal regions of antifreeze protein and green fluorescent protein in Bacillus subtilis on their secretion. The results show that the antifreeze protein is difficult to be detected by SDS-PAGE, and it is difficult to detect low molecular weight type I AFP by western blotting. The green fluorescent protein expressed in high amount in the total cell lysate of Bacillus subtilis. And the expression level of the green fluorescent protein with histidine-tag is higher. In vitro protease assay shows that the green fluorescent protein with histidine-tag is more resistant to protease than the GFP without histidine-tag. Histidine-tag is not the main factor to block green fluorescent protein secretiion in Bacillus subtilis DB430.