Enhancement of anti-HIV peptide T-20 production in recombinant E. coli by analysis of metabolic load = 대사분석을 이용한 재조합 대장균에서의 항 HIV 펩타이드 T-20의 생산 증대

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Anti-HIV peptide T-20 is a polar peptides consists of 36 amino acids corresponding to residues 127-162 in the transmembrane segment of the HIV envelope glycoprotein (gp41) This peptide targets the fusion phase of HIV infection, and is considered to interfere with the conformational changes in gp41 to prevent fusion of viral and host cell membranes. T-20 is the most complex synthetic peptide ever chemically manufactured at a large scale, requiring an unprecedented complexity of manufacturing process that involves 106 production steps, as opposed to 8-10 steps, for typical synthetic process. Therefore, biologic production could be a good alternative solution to the chemical one for mass production of T-20 For production of foreign protein and peptides, medium optimization, strong promoter, and high copy number plasmid were routinely used. These strong expression system can increase the expression of target protein, but it causes serious metabolic load to host cell. Under metabolic load, stringent response occurs. Stringent response is a complicated prokaryotic response system regulates gene expression in transcription level to survive in nutrient depleted condition. On this response, cell stops the producing of foreign proteins and switches the gene expression to its survival. This means protein production machinery of host cell is not fully utilized for enough time. Guanosine tetraphosphate (ppGpp) is the signal and effector molecule of this response. By monitoring the intracellular ppGpp level, the occurrence of stringent response and metabolic load can be determined. Fed batch fermentation was performed for T-20 mass production with E.coli BL21/pET23a-G3T20 which has isopropyl-_-D-thiogalactopyranoside (IPTG) inducible expression system. During the fermentation, intracellular ppGpp was measured, and ppGpp was increased from 1.55 umol/gDCW to 4.5 umol/gDCW in a hour after induction. Also cell growth retardation was observed. It might be that induction of tar...
Kim, Jung Hoeresearcher김정회researcher
한국과학기술원 : 생명과학과,
Issue Date
268696/325007  / 000985831

학위논문(박사) - 한국과학기술원 : 생명과학과, 2007. 8, [ viii, 90 p. ]


anti-hiv; T-20; production; metabolic load; proteomics; 항HIV; T-20; 생산; 대사부하; 단백질체학; anti-hiv; T-20; production; metabolic load; proteomics; 항HIV; T-20; 생산; 대사부하; 단백질체학

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