Cloning and expression of N-terminal first-half extracellular domain of rat iGluR1 = 쥐의 이온 수송 글루타메이트 수용체의 N-말단 부위의 클로닝과 대장균에서의 발현

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N-terminal first-half extracellular domain of ionotropic glutamate receptor subunit 1 (iGluR1N), without any specified function yet, was cloned from cDNA library of rat brain by PCR. iGluR1N protein was stably expressed with its native signal sequence in E.coli under Ptac promoter to form insoluble aggregate. In order to export this protein into the periplasm, the signal sequence was replaced by the signal sequence of periplasmic ribose-binding protein (RBP) of E.coli. The signal sequence of RBP-iGluR1N fusion protein was processed but the mature part of iGluR1N stayed at membrane giving lethal effect to the cell. It seems that the lethal effect by the fusion protein results from the jamming of secretory machinery leading to accumulation of precursor proteins inside the cytoplasm. The expression of iGluR1N also gave lethal effect to the cell. A spontaneous mutant that could survive with iGluR1N expression was selected. In this suppressor mutant, the amount of expressed iGluR1N did not decrease still forming aggregate. The suppressor mutant grew slower than wild type and the suppressor cells were shown to be filamentous form at stationary phase. The spontaneous mutation of this suppressor was mapped at 2 min region of E.coli chromosome. The suppressor mutant could not relieve the lethality by RBP-iGluR1N expression, which indicated that the modes of lethality resulted from the expression of two proteins were quite different.
Park, Chan-KyuresearcherChung, Jong-Kyeongresearcher박찬규researcher정종경researcher
한국과학기술원 : 생물과학과,
Issue Date
112657/325007 / 000953282

학위논문(석사) - 한국과학기술원 : 생물과학과, 1997.2, [ iv, 48 p. ]


Translocation; Protein folding; 글루타메이트 수용체; 전위 신호 배열; 전위; 단백질 폴딩; Glutamate receptor; Signal sequence

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