• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.750-753
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• 2001

Proteomics is a formalized approach for obtaining a rapid snap-shot of the protein complement of a tissue, cell or cell component. Such an approach is powerful in that it allows a parallel assessment of temporal protein fluxes. This is an important concept in view of the dynamic nature of protein expression. Undoubtedly, changes in protein expression are essential in any study aimed at investigating cellular networks. In this study, we analyzed and compared the proteomes of recombinant E. coli strain before and after induction. Proteome expression patterns of recombinant E. coli were resolved on 2D-gels, and the variations in the relative expression level of particular proteins were examined using software-aided protein quantification tool. We observed above 800 spots on a 2D-gel using Melanie II software. Many proteins which involved in chaperones were significantly up-regulated in recombinant E. coli. Therefore, it could be concluded that the expression of recombinant protein in E. coli acted as a stress to the cells, which change cells ability to synthesize proteins and induced the expression of various protective proteins.

• Journal of Microbiology and Biotechnology
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• 제15권2호
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• pp.403-411
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• 2005

The relationship between plasmid (~9.5 kb) and pediocin PA-1 in P. acidilactici K10 was confirmed by plasmid curing. The pediocin operon of P. acidilactici K10 was amplified by PCR (polymerase chain reaction), and the nucleotide sequence was analyzed. The sequence of the pediocin operon of P. acidilactici K10 was similar to those of P. acidilactici strains producing pediocin PA-1/ AcH. For the expression of pediocin PA-1 in E. coli, a pQEPED (pQE-30 Xa::mature pedA) was constructed. His-tagged recombinant pediocin PA-1 (-6.5 kDa) was translated by cell-free in vitro transcription and translation using pQEPED as a DNA template. Theresult of slot blotting assay showed that transcription of recombinant pedA in E. coli M15 was induced by the addition of isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) at the final concentration of 1 mM. Although the recombinant pediocin PA-1 inhibited the growth of E. coli, it was expressed in the host strain and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under denaturing condition. This is the first report for the production and one-step purification of biologically active recombinant pediocin PA-1 in E. coli.

• International Journal of Industrial Entomology and Biomaterials
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• 제17권1호
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• pp.137-141
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• 2008

We previously isolated and cloned a cDNA of abaecin from the Bombus ignitus. In an effort to produce a large amount of soluble abaecin at low cost, we successfully expressed the peptide in Escherichia coli that are highly sensitive to its mature form. For this, we fused the peptide encoding 39 amino acids of mature B. ignitus abaecin to the thioredoxin gene together with a C-terminal 6xHis tag. An enterokinase cleavage site was introduced between the 6xHis tag and mature abaecin to allow final release of the recombinant peptide. A high yield of 9.6 mg soluble fusion protein from 200 ml of bacterial culture was purified by $Ni^{2+}$-charged His-Bind resin affinity column, and 1.4 mg of pure active recombinant abaecin was readily obtained by enterokinase cleavage, followed by affinity chromatograph. The molecular mass of recombinant abaecin peptide was determined by Tricin-SDS-PAGE analysis. The recombinant abaecin exhibited antibacterial activity against Gram-negative bacteria.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1726-1736
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• 2013

Biosynthesis of indole-3-acetic acid (IAA) via the indole-3-pyruvic acid pathway involves three kinds of enzymes; aminotransferase encoded by aspC, indole-3-pyruvic acid decarboxylase encoded by ipdC, and indole-3-acetic acid dehydrogenase encoded by iad1. The ipdC from Enterobacter cloacae ATCC 13047, aspC from Escherichia coli, and iad1 from Ustilago maydis were cloned and expressed under the control of the tac and sod promoters in E. coli. According to SDS-PAGE and enzyme activity, IpdC and Iad1 showed good expression under the control of $P_{tac}$, whereas AspC was efficiently expressed by $P_{sod}$ originating from Corynebacterium glutamicum. The activities of IpdC, AspC, and Iad1 from the crude extracts of recombinant E. coli Top 10 were 215.6, 5.7, and 272.1 nmol/min/mg-protein, respectively. The recombinant E. coli $DH5{\alpha}$ expressing IpdC, AspC, and Iad1 produced about 1.1 g/l of IAA and 0.13 g/l of tryptophol (TOL) after 48 h of cultivation in LB medium with 2 g/l tryptophan. To improve IAA production, a tnaA gene mediating indole formation from tryptophan was deleted. As a result, E. coli IAA68 with expression of the three genes produced 1.8 g/l of IAA, which is a 1.6-fold increase compared with wild-type $DH5{\alpha}$ harboring the same plasmids. Moreover, the complete conversion of tryptophan to IAA was achieved by E. coli IAA68. Finally, E. coli IAA68 produced 3.0 g/l of IAA after 24 h cultivation in LB medium supplemented with 4 g/l of tryptophan.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권1호
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• pp.13-16
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• 2000

Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed in Escberichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced in E. coli cytoplasm were easily dissolved by simple alkaline pH shift $(8\rightarrow12\rightarrow8)$. Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.

• 생명과학회지
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• 제25권12호
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• pp.1339-1346
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• 2015

본 연구는 카로티노이드 생합성 유전자군이 형질전환된 Escherichia coli에서 archaea chaperonin을 공발현 시킴으로 카로티노이드의 생산량을 증대시키는 것이 목표이다. 카로티노이드는 식물, 박테이라, 조류 등이 생합성하는 노란색, 오렌지색, 붉은색 계통의 색소로 이들은 식품 또는 양식 사료로 주로 이용되는 물질이다. 본 연구자들은 선행연구를 통하여 Paracoccus haeundaensis로부터 카로티노이드 유전자군을 cloning하였고 이들 유전자군의 생화학 및 효소학적 기능성을 분석하는 연구 결과와 카로티노이드 생합성 유전자군(crtE, crtB, crtI, crtY, crtZ, crtW, crtX)을 대장균에 형질전환하여 400 μg/g dry cell weight (DCW)의 아스타잔틴을 생산하는 연구 결과를 보고 하였다. 본 연구에서는 이들 유전자군과 archaea chaperonin을 공발현시켜 대장균에서 astaxanthin을 890 μg/g dry cell weight (DCW)로 생산하였으며, 이는 선행 연구된 결과 보다 약 2배 이상의 astaxanthin 생산량을 향상 시키는 연구 결과이다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2001년도 추계학술발표대회
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• pp.711-714
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• 2001

본 연구에서는 말라리아 항원의 생물학적 대량 생산 공정을 디자인하기 위하여 자체 개발한 재조합 E. coli 시스템의 여러 가지 조업 조건들, 즉 균체 성장과 외래 단백질의 유도 발현에 영향을 미치는 초기 배지 pH, 유도 발현 이후의 조업 온도 및 타이밍 , 그러고 기간 등을 조사함과 동시에 최적 배양 전략을 탐색하였다.

• 미생물학회지
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• 제32권1호
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• pp.28-33
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• 1994

정상적인 세포에서 tau 단백질은 신경세포의 축색돌기에 있는 미세소관(microtubule)과 결합하고 있지만, Alzheimer병 세포의 경우 그 단백질은 몇몇 신경세포의 체세포 수지상조직(somatodendrite) 부위에 고착되어서 이중나선 섬유(paired helical filament; PHF)의 주성분을 이루게 된다. 따라서 뇌에PHF가 축적되는 특성 파악과 그들을 야기시키는 요인분석의 일환으로 다량의 순수한 tau 단백질을 확보하기 위하여 본 연구에서는 인체 tau 유전자의 cDNA를 클로닝하고 Escherichia coli에서의 발현을 유도하였다.

• 한국미생물·생명공학회지
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• 제23권5호
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• pp.550-555
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• 1995

A strain of Pseudomonas sp. isolated from soil was shown to produce a high level of extracellular endo-inulinase. In this work, the endo-inulinase gene (inu1) of the bacterial strain was cloned into the plasmid pBR322 by using EcoRI restriction endonuclease and E. coli HB101 as a host strain. One out of 7, 000 transformants obtained from the above cloning experiment formed a clear zone around its colony on the selective medium supplemented with 2.0% inulin after a prolonged incubation at 37$\circ$C and subsequent cold shock treatment. The functional clone was found to carry a recombinant plasmid (pKMG50) with a 3.7 kb genomic insert containing the genetic information for the inulinase activity. The inulinase from E. coli HB101/pKMG50 was proved to be an endo-acting enzyme and produced constitutively in the recombinant E. coli cells. Zymogram of the enzyme from the recombinant cells with inulin substrate indicated that the molecular mass of the active protein was 190 Kd, while that of the endo-inulinase from the Pseudomonas strain was 170 Kd. This size discrepancy suggested that the inulinase from the recombinant E. coli HB101 cells might be the initial product of translation, not the mature form produced in the strain of Pseudomonas sp..

• Journal of Microbiology and Biotechnology
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• 제17권6호
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• pp.1045-1048
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• 2007

Two genes, dps encoding decaprenyl diphosphate synthase and dxs encoding 1-deoxy-D-xylulose 5-phosphate synthase, were isolated from Rhizobium radiobacter ATCC 4718. DNA sequencing analysis of the dps and dxs genes revealed an open reading frame of 1,077 bp and 1,920 bp, respectively. The heterologous expression in Escherichia coli BL21(DE3) was carried out in order to identify their functions. Recombinant E. coli BL21(DE3) harboring the dps gene produced $CoQ_{10}$ as well as $CoQ_8$ and $CoQ_9$, whereas E. coli harboring only the dxs gene produced more $CoQ_8$ compared with the wild-type E. coli. Additionally, the coexpression of dps and dxs genes in E. coli was carried out. The recombinant E. coli harboring only the dps gene produced $0.21{\pm}0.04\;mg/l$ of $CoQ_{10}$, whereas the coexpressed E. coli with dps and dxs genes produced $0.37{\pm}0.07\;mg/l$ of $CoQ_{10}$. HPLC analysis also showed that the $CoQ_{10}$ fraction (100% of the total CoQs distribution) was increased from $15.86{\pm}0.66%$ (only dps) to $29.78{\pm}1.80%$ (dps and dxs).