초록 ▼

의약용 재조합 단백질 및 산업용 효소의 대량생산을 위하여 사용되고있는 고가의 유도물질(예: IPTG 등)의 첨가가 필요 없는, 즉 발현유도과정이 없는 항시적 고발현 시스템(high-level constitutive expression system, HCE system)을 이용하여 진단용 효소인 HAO(Histamine oxidase), 산업용 생물촉매인 TPL(Tyrosine phenol-lyase), TNA(Tryptophane indole-lyase), D-AAT(D-Amino acid aminotransferase) 및 항암제

의약용 재조합 단백질 및 산업용 효소의 대량생산을 위하여 사용되고있는 고가의 유도물질(예: IPTG 등)의 첨가가 필요 없는, 즉 발현유도과정이 없는 항시적 고발현 시스템(high-level constitutive expression system, HCE system)을 이용하여 진단용 효소인 HAO(Histamine oxidase), 산업용 생물촉매인 TPL(Tyrosine phenol-lyase), TNA(Tryptophane indole-lyase), D-AAT(D-Amino acid aminotransferase) 및 항암제 및 면역조절제로 사용되는 의약용 재조합 단백질(Tumor necrosis factor-$\alpha$ TNF-$\alpha$), Granulocyte-colony stimulating factor (G-CSF), Vesicular endotherial growth factor(VEGF), Interleukine-6 (IL-6))의 대량 생산을 위한 항시적 고발현시스템 확립 및 청정 대량생산을 위한 항생제 비의존성 항시적 고발현 시스템(pHCE-AMPFREE)개발과 이를 이용한 새로운 방식의 재조합 단백질 청정 대량생산기술의 개발(산엄균주 E.coli WM334, 플라스미드 안정성 100%, 발현율 30%이상, 건조균체 중량 15g/L)을 통하여 생산원가를 획기적으로 절감하고 안정적인 조업이 가능한 새로운 방식의 재조합 단백질 대량생산기술을 확립함

Abstract ▼

The high-level constitutive expression (HCE) system allows the high constitutive expression of recombinant protein using E. coli and B. subtilis as the host organisms. Expressed target protein constitutes more than 20 to 70% of total proteins of recombinant E. coli showing high expression efficiency

The high-level constitutive expression (HCE) system allows the high constitutive expression of recombinant protein using E. coli and B. subtilis as the host organisms. Expressed target protein constitutes more than 20 to 70% of total proteins of recombinant E. coli showing high expression efficiency. Since protein is expressed constitutively, there is no need to add inducers, and production cost can be minimized. B. subtilis HCE system allows constitutive expression of recombinant protein using B. subtilis chungkookjang strain as the host. This host organism was isolated from Chungkookjang Korean traditional soybean food, and is very safe and designated as a GRAS microorganism. The expression vectors are constructed to add a secretion signal sequence of Lipase, YncM and Subtilisn E to the N-terminal region of the target protein; the expressed recombinant protein is therefore secreted into culture medium.
The two HCE systems are promising for industrial production of proteins used for foods or medicines, as production cost, microorganism culture and scale up are convenient. Both expression systems are very unique, and in terms of cost and safety, highly suitable for industrialization. For the mass production of industrial enzymes (Histamine oxidase, Tyrosine phenol-lyase, Tryptophan indole-lyase, D-amino acid aminotransferase) and medicinal proteins (human tumor necrosis factor-$\alpha$, human granulocyte colony stimulating factor, human vesicular endotherial growth factor, human interleukin-6) using the HCE systems, expression systems of each recombinant protein were constructed and the constitutive expression rate of them in total proteins were followed : HAO ; 10%, TPL ; 35%, TNA 50%, D-AAT ; 40%, TNF-a ; 50%, G-CSF ; 10%, VEGF 10%, IL-6 ; 5%.
In order to develop efficient expression systems for the industrial production of useful enzyme and protein, HCE-AMPFREE system using the constitutively expressing HCE promoter and amino acid auxotrophy was costructed. The HCE-AMPFREE system is not required to add IPTG for induction and antibiotics for the selection of recombinants and the maintenance of plasmids. As culture scale is increased, the use of antibiotics and inducers leads to the higher cost and the environmental pollution. Thus, productivities of recombinant proteins (TPL, TNA) with HCE-AMPFREE system was came to 30% of expression efficiecncy and 15g/L of dry cell weight (DCW) of the host cells.
The cost-effective expression of recombinant protein is one of the most important in overall process to production of recombinant proteins and enzymes. The expression efficiency of recombinant TNF-$\alpha$ using pHCE in E.coli was compared with that of pET-14b vector which is under control of T7 promoter induced by IPTG. To compare the efficiency for the foreign gene expression, the TNF-$\alpha$ was expressed by either pHCE or pET vector. The TNF-a was recovered mostly in soluble fraction, the expression level was more than 25% of total soluble proteins of recombinant E. coli, and its biological activity was confirmed by ELISA and L929 cytotoxicity assay. Total amount of TNF-$\alpha$ produced by pHCE system was approximately twice of that produced by pET system under the optimal fermentation conditions. The economical efficiency of the produced TNF-a by the pHCE system is more fourteen times than the produced TNF-$\alpha$ by the inducible expression system.
Accordingly, the results of the current our study indicate that the technology of recombinant protein production using pHCE system is more convenient and economical than the inducible pET system for the mass production of recombinant proteins.

목차 Contents

• 제 1 장 연구개발과제의 개요...14
• 제 1 절 연구개발의 배경 및 필요성...14
• 1. 연구개발배경...14
• 2. 연구개발의 필요성...16
• 제 2 절 연구개발의 경제,사회,기술적 필요성...18
• 1. 기술적 측면...18
• 2. 경제, 산업적 측면...19
• 제 2 장 국내외 기술개발현황...20
• 제 1 절 국내의 기술개발 현황...20
• 제 2 절 현 기술상태의 취향성 및 전망...22
• 제 3 절 국내외 관련분야의 환경변화...23
• 1. 재조합 의약용단백질의 대량생산기술 연구사례...23
• 2. 재조합 의약용단백질의 생산관련기술...24
• 3. 재조합 의약용단백질의 국외시장 현황 및 전망...24
• 제 3 장 연구개발수행 내용 및 결과...27
• 제 1 절 연구개발 수행 내용...28
• 제 2 절 연구개발 결과...30
• 1. 재조합 단백질 대량생산 시스템의 확립...30
• 2, 재조합 의약용 단백질의 가용성발현을 위한 항시적 고발현 기술 구축...60
• 3. 항시적 고발현 기술 이용 재조합 단백질 생산의 경제성 분석...81
• 4. 항시적 고발현 기술 이용 재조합 의약용 단백질의 생물활성 검증 결과...86
• 제 4 장 목표달성도 및 관련분야에의기여도...87
• 제 1 절 연구개발목표의 달성도...87
• 제 2 절 연구개발 관련분야의 기여도...90
• 제 5 장 연구개발결과의 활용계획...91
• 제 1 절 활용계획...91
• 제 2 절 재조합단백질 수탁생산 사업 활용...92
• 1. 재조합단백질 수탁생산 사업의 배경...92
• 2. 항시적 고발현기술 이용 재조합단백질 수탁생산 사업으로의 활용계획...93