| Summary: | 碩士 === 國立虎尾科技大學 === 生物科技研究所 === 98 === A lot of proteins could be extracted from the jelly curd. Some pericarpial proteins extracted from the jelly curd have been identified as a pectin methylesterase (PME), chitinase and thaumatin-like protein (TLP). The MALDI-TOF analysis revealed that the protein extract of jelly fig achenes resolved in SDS-PAGE may also contain other PR proteins, such as PR-4 and PR-1. PR-4 was classified into two groups, Class I and II. Class I and II shared highly sequence similarity. However, Class I of the PR-4 contains extra chitin binding domain. Degenerate primers were designed according to the conserved sequence of known PR-4, and cDNA fragment encoding jelly fig PR4 (FaPR4) was cloned by PCR. FaPR4 comprises 916 bp with open reading frame of 618 bp, which encodes 205 a.a.. The predicted mature molecular mass of FaPR4 is 19.8 kDa. FaPR4 was expressed in E. coli as insoluble protein and its antibodies against FaPR4 were prepared. FaPR4 would be detected in the protein extract of jelly fig achenes by Western blot analysis. Furthermore, r-FaPR4, r-FaPR4(No) and r-FaPR4(No/mutant) were expressed in yeast as secreted proteins. r-FaPR4 is the recombinant FaPR4, r-FaPR4(No) is the recombinant FaPR4 without chitin binding domain, and r-FaPR4(No/mutant) is r-FaPR4(No) containing one amino acid mutant. Native FaPR4 (partial purify), r-FaPR4 and r-FaPR4(No) exhibited RNase activity; while r-FaPR4(No/mutant) showed no RNase activity. Moreover, r-FaPR4 shows thermal stability.
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