초록 ▼

- 재조합 대장균을 사용한 5L 발효조에서 확립된 최적 fed-batch 발효 공정을 50L로 scale-up하고 컴퓨터 control feeding program을 이용한 유가식 고농도 배양공정개발
- Ferritin 정제시 2단계 세포파쇄를 이용한 전처리 및 실리카 분말을 사용한 흡착/탈착공정으로 단백성 불순물을 효과적으로 제거하는 정제공정 확립
- 재조합 대장균에서 발현된 FerH와 FerL을 정제한 후, in vitro에서 혼합, assembling을 유도함으로써 Human 페리틴과 가장 유사한 페리틴 합성기술 개

- 재조합 대장균을 사용한 5L 발효조에서 확립된 최적 fed-batch 발효 공정을 50L로 scale-up하고 컴퓨터 control feeding program을 이용한 유가식 고농도 배양공정개발
- Ferritin 정제시 2단계 세포파쇄를 이용한 전처리 및 실리카 분말을 사용한 흡착/탈착공정으로 단백성 불순물을 효과적으로 제거하는 정제공정 확립
- 재조합 대장균에서 발현된 FerH와 FerL을 정제한 후, in vitro에서 혼합, assembling을 유도함으로써 Human 페리틴과 가장 유사한 페리틴 합성기술 개발
- Refolding이 이루어지기 전에 Iron을 첨가하였을 때 철분이 함유된 페리틴이 형성되는 것을 Prussian blue stain으로 확인하였으며, FerH만 존재하였을 때보다 FerL이 15% ~ 35% 정도 함유되어 있을 때 철 함량이 높은 것을 확인
- 효모 S. cerevisiae를 사용한 재조합 ferritin 생산 기술 개발 및 최적 발효조건 확립
: Kozak sequence 조절에 의한 발현 최적화
: Codon Usage 최적화에 의한 발현 최적화

Abstract ▼

Human ferritin naturally forms a spherical copolymer consisting of 24 subunits of Hand L-chains and the two chains have cooperative role in the iron uptake mechanism of human ferritin. We developed a recombinant ferritin H/L-hybrid by placing H-chain subunit directly at the N-terminus of L-chain fer

Human ferritin naturally forms a spherical copolymer consisting of 24 subunits of Hand L-chains and the two chains have cooperative role in the iron uptake mechanism of human ferritin. We developed a recombinant ferritin H/L-hybrid by placing H-chain subunit directly at the N-terminus of L-chain ferritin and expressed it using T7 promoter system in E. coli. The soluble ferritin H/L-hybrid produced was biologically active with the iron storage capacity equivalent to ferritin standard. Over-expressed recombinant ferritin in Escherichia coli formed inclusion bodies through noncovalent molecular interaction and easily dissolved with regaining the iron-uptake activity by a simple pH-shift process at high protein concentration (>600 mg/L). Ferritin was relatively thermostable at low protein concentration (0.2 g 1-1), but it became extremely thermolabile at high protein concentration (1.3 g/L), i.e., more than 80% of ferritin was co precipitated within 5 min under the same heat-induced denaturation condition. Aggregation rate constant for initial 5 min at high protein concentration was 6.04 ${\times}$ 10-3 $s^{-1}$ for ferritin. Glucagon ferritin mutant, consisting of an N-terminus fusion partner, human glucagon (29-residue a-helical peptide), showed significantly enhanced thermal stability even at high protein concentration. That is, in spite of 40-min heat treatment, more than 50% of glucagon -ferritin the still remained soluble with maintaining the same functional properties. The aggregation rate constants were 2.75 ${\times}$ 10-4 and 2.80 ${\times}$ 10-4 s-1 at low and high concentration, respectively, for glucagon-ferritin. These results suggest a critical participation of the N-terminal domain of ferritin in the temperature-induced aggregation pathway. Presumably, partially denatured amino terminus of ferritin is involved in nonspecific molecular interaction resulting in the off-pathway aggregation. It is notable that the purified glucagon-ferritin showed the same molar capacity of iron (Fe+3) storage as standard ferritin. From the analysis of fluorescence emission spectrum, the physical stability of glucagon-ferritin was also very comparable to that of standard ferritin under the various denaturation conditions induced by GdnHCl. The expression vectors harboring human ferritin gene were constructed for the s. cerevisiae, P. pastoris, H. polymorpha. To enhance the expression level of ferritin, kozak sequence and codon usage were optimized. The final ferritin concentrations were achieved 2.5g/L in the fed batch fermentation using high cell density culture mode. Soluble form of fusion ferritin expressed in recombinant E. coli was separated from the E. coli cells disrupted by two-step sonications, followed by silica powder adsorption. Ferritin was recovered from the silica powder by distilled water, which was applied to gel filtration chromatography (GFC). Collected ferritin fractions from the GFC were assayed via iron-uptake and molecular weight determined using GF-HPLC. Fused ferritin showed a higher activity than the H- or L- chain by two times. SDS-gel electrophoresis showed a single band of 38 kDa with heavy and light chains. MALDI-TOF MS gave a molecular weight of fusion ferritin was 38 kDa. The specific activity and yield of purified fusion ferritin are 0.41 $Fe^{3+}$ mg $mg^{-1}$ of protein and 66%. Those values are larger than the previous ones of 0.2 $Fe^{3+}$ mg $mg^{-1}$

목차 Contents

• 표지...1
• 제출문...2
• 보고서 초록...3
• 요약문...4
• SUMMARY...6
• CONTENTS...8
• 목차...9
• 제1장 연구개발과제의 개요...10
• 제1절 연구개발의 목적...10
• 제2절 연구 개발의 필요성 및 범위...10
• 제2장 국내외 기술개발 현황...13
• 제3장 연구개발수행 내용 및 결과...14
• 제1절 연구내용...14
• 제2절 연구결과...15
• 제3절 결론...40
• 제4장 목표달성도 및 관련분야에의 기여도...42
• 제1절 목표달성도...42
• 제2절 관련분야에의 기여도...42
• 제5장 연구개발결과의 활용계획...43
• 제6장 연구개발과정에서 수집한 해외과학기술정보...44
• 제7장 참고문헌...45
• 위탁연구과제(I)...47
• 위탁연구과제(II)...93