• Journal of Microbiology and Biotechnology
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• 제3권3호
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• pp.161-167
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• 1993

Immobilized metal ion affinity chromatography (IMAC) was used to separate various types of recombinant hirudins from the culture broth. The wild type hirudin exhibited a retention in Cu(II)-chelated affinity chromatgoraphy since it contained a single exposed histidine at position 51. To obtain a stronger retention on an IDA-Cu(II) column, the hirudin variants were genetically engineered to contain one or two histidine (s) more than the wild type. While the affinity of the variants for IDA-Cu(II) ligand increased in comparison to that of the wild type, the antithrombin activities reduced to a certain degree. Cu(II), Ni(II) and Zn(II) ions were applied separately to the metal chelate column to investigate ligand specificity with respect to protein retention. As a result, the Cu(II) chelated chromatography gave the best resolution for all the hirudins tested and appeared to be the only IMAC that could be used generally for the purification of hirudins with a decreasing pH gradient.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.538-538
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• 2002

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.344-349
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• 2004

A gene encoding inulin-degrading enzyme of Bacillus sp. snu-7 with ORF of 1536 nucleotides was cloned. And it was overexpressed as His-tagged protein in E. coli BL21(DE3) pLysS using pRSET B vector containing mature enzyme sequence. Maximum enzyme production was achieved by IPTG (0.1 mM) induction at $OD_{600}$ 1.2 and $30^{\circ}C$ followed by 6 h incubation. The expressed protein purified through immobilized metal affinity chromatography showed molecular mass of 60 kDa on SDS-PAGE. Results of thin-layer chromatography using inulin as a substrate showed the enzyme to be an exotype inulinase capable of producing only monomeric fructose as a product. $K_m$ and $k_{cat}$, for the hydrolyses of inulin and sucrose were $2.28\pm0.08$ mM and 358.05$\pm$20.38 $min^{-l}$, and 22.02$\pm$0.41 mM and 4619.11$\pm$215.12 $$min^{-1}, respectively. Optimal activity of the exoinulinase occurred at pH 7.0 and $50^{\circ}C$.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.533-533
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• 2003

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.539-542
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• 2003

SDS-PAGE 결과 elution pH에 따라 분리되는 band pattern은 비슷하게 2band의 양상을 보이지만, FI값을 비교하여 보았을 때 다른 pH보다 8.0에서 가장 높은 수준을 보였으므로 GFPuv/cytochrome c-552 fusion Protein의 분리 ${\cdot}$ 정제에 가장 적합한 pH를 8.0으로 정하였다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권2호
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• pp.144-149
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• 2001

For the production and purification of a single chain human insulin precursor, four types of fusion peptides $\beta$-galactosidase (LacZ), maltose binding protein (MBP), glutathione-S-transferase (GST), and (His)(sub)6-tagged sequence (HTS) were investigated. Recombinant E. coli harboring hybrid genes was cultivated at 37$\^{C}$ for 1h, and gene induction occurred when 0.2mM of isopropyl-D-thiogalactoside (IPTG) was added to the culture broth, except for E. coli BL21 (DE3) pLysS harboring a pET-BA cultivation with 1.0mM IPTG, followed by a longer than 4h batch fermentation respectively. DEAE-Sphacel and Sephadex G-200 gel filtration chromatography, amylose affinity chromatography, glutathione-sepharose 4B affinity chromatography, and a nickel chelating affinity chromatography system as a kind of immobilized metal ion affinity chromatography (IMAC) were all employed for the purification of a single chain human insulin precursor. The recovery yields of the HTS-fused, GST-fused, MBP-fused, and LacZ-fused single chain human insulin precursors resulted in 47%, 20%, 20%, and 18% as the total protein amounts respectively. These results show that a higher recovery yield of the finally purified recombinant peptides was achieved when affinity column chromatography was employed and when the fused peptide had a smaller molecular weight. In addition the pET expression system gave the highest productivity of a fused insulin precursor due to a two-step regulation of the gene expression, and the HTS-fused system provided the highest recovery of a fused insulin precursor based on a simple and specific separation using the IMAC technique.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.612-612
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• 2003

• Bulletin of the Korean Chemical Society
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• 제28권9호
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• pp.1549-1552
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• 2007

The cytoplasmic domain of syndecan-4, a type I transmembrane heparan sulfate proteoglycan, was overexpressed as a fused form with the ubiquitin molecule in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography (IMAC). The cytoplasmic domain was released from its fusion partner by using yeast ubiquitin hydrolase (YUH), and subsequently purified by reverse phase chromatography. The integrity of the resulting peptide fragment was checked by MALDI-TOF and NMR spectroscopy. The yield of the peptide was 3.0-1.5 mg per liter in LB or minimal medium, respectively. The recombinant expression and purification of this domain will enable us its structural and functional studies using multidimensional NMR spectroscopy.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권6호
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• pp.402-409
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• 2001

Different bioligands carrying synthetic adsorbents have been reported in the literature for protein separation, We have developed a novel and new approach to obtain high protein ad-sorption capacity utilizing 2-methacrylamidohistidine(MAH) as a bioligand. MAH was synthe-sized by reacting methacrylocholride and histidine, Spherical beads with an average size of 150-200㎛ were obtained by the radical suspension polymerization of MAH and 2-hydrosyethyl-methacrylate(HEMA) conducted in an aqueous dispersion medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6㎡/g . Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental analysis. Then Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g.These affinity beads with a swelling ration of 65% and containing, 1.6 mmol MAH/g were used in the adsorption/desorption of human serum albumin(HSA) from both aqueous solutions and hu-man serum. The adsorption of HSA onto p(HEM-co-MAH) was low(8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA ad-sorption ws observed at pH 8.0 Higher HSA adsorption was observed from human plasma(94.6 mgHSA/g) Adsorption of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5mg/g for γ-globulin. The total protein adsorption was determined as 107.1mg/g. Desorption of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5 M NaSCN, High desorption rations(up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities.

• 한국산학기술학회논문지
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• 제10권6호
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• pp.1287-1291
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• 2009

시안화물을 포름아미드로 변환시키는 nitrilase의 일종인 시안 수화효소 (cyanide hydratase, CHT) 를 진균류인 Neurospora crassa 와 Aspergillus nidulans로부터 유전자 조작을 통하여 His에 태그 또는 언태그된 형태로 대장균에 형질변환시켜 발현하였다. 발현된 효소를 고정 metal affinity chromatography로 정제하였다. 정제된 효소들의 pH 안정성, 동력학적 매개변수의 값을 검토하였다. 실험 결과 N. crassa 의 CHT가 50% 정도 더 넓은 pH 안정 범위를 가졌고 3배 가량 turnover rate도 높았다. 반면 A. nidulans CHT의 Km 값 (효소포화 용량)이 N. crassa CHT보다 더 크게 나타났다. 두 진균류에서 CHT의 유도발현은 질소성분과 상관없이 KCN에 의해 가능하였으며, 생분해 실험결과 N. crassa CHT에 의해 최대 82%/h의 시안분해가 가능하였다.