1994 Fiscal Year Final Research Report Summary
Structure and Function of Heat-stable Sweet Protein, Mabinlin
| Project/Area Number |
05044126
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Yokohama National University |
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Principal Investigator |
KURIHARA Yoshie Faculty of Education Yokohama National University, 教育学部, 教授 (90017715)
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| Co-Investigator(Kenkyū-buntansha) |
りゅう 小ずう 昆明植物研究所, 研究員
胡 忠 昆明植物研究所, 教授
KATAHIRA Masato Faculty of Engineering Yokohama National University, 工学部, 講師 (70211844)
MASUDA Yutaka School of Pharmaceutical Sciences Showa University, 薬学部, 助手 (10255862)
NAKAYA Kazuyasu School of Pharmaceutical Sciences Showa University, 薬学部, 教授 (40053855)
HU Zhong Kunming Institute of Botany The Academy of Sciences of China
LIU Xiaozhu Kunming Institute of Botany The Academy of Sciences of China
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| Project Period (FY) |
1993 – 1994
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| Keywords | Sweet protein / heat-stable protein / mabinlin / cDNA cloning |
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| Research Abstract |
Total RNA was extracted by the phenol-SDS method from the seeds of Capparis masaikai Levl. Poly (A) ^+RNA was purified by Oligotex-dT30 Super. Double-stranded cDNA was synthesized using a cDNA synthesis kit. lambdagt10 cDNA library was constructed with Complete rapid cloning system-lambdagt10. Two oligonucleotide primers, 5'-AGCATATGCA (AG) (TC) TITGG (CA) GITG (TC) CA-3' and 5'-CGGATCCCTACCAIGCIC (GT) AGAAIGG (AG) C-3', were prepared on the basis of the amino acid sequences QLWRCQ and CPFRAW,respectively. The PCR was conducted using the cDNA from seeds with 30 cycles of 1 min at 96 ﾟC for denaturation, 1 min at 50ﾟC for annealing, and 1 min at 72ﾟC for extension. The amplified DNA fragment was used as a probe to screen the cDNA library. Recombinant phages were screened by plaque hybridization with the ^<32>P-labeled probe. We finally obtained 5 clones out of 1.0x10^5 plaques. One clone with a cDNA insert of 630 bp was subjected to nucleotide sequencing. The clone contained single open reading frame of 465 bp. The amino acid sequence determined in a previous study matched the deduced sequence from 36 to 68 and from 85 to 155. The N-terminal extension of 20 amino acids (Met^1 to Ala^<20>) is rich in hydrophobic amino acid and appears to be a signal sequence.
The DNA encoding mabinlin II was inserted into the expression vector, pET15b. This expression vector termed pMAB2 contained bacteriophage T7 promoter. The vector was used to transform E.coli strain BL21. E.coli BL21/pMAB2 was induced by isopropylthio-beta-D-galactoside (IPTG) in LB medium. The protein with an apparent molecular weight (15kDa) identical to that of mabinlin II protein was detected by SDS-PAGE of whole cell lysates. Western blotting analysis showed that this induced protein reacted with anti-mabinlin II antibody.
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