• KSBB Journal
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• v.15 no.3
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• pp.238-242
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• 2000

A fusion protein of human interleukin-2(hiL-2) and glucagon which was expressed in Escherichia coli. was digested with enterokinase for recovery of hIL-2 from the fused protein. To obtain hIL-2 of optimum recovery hydrolysis reaction were performed under various conditions of urea additives and reaction time. hIL-2 was finally purified by RP-HPLC(reversed phase-HPLC) to remove cleaved G3 fusion partner and residual uncleaved G3-IL-2 HIL-2 was eluted in a single peak at 100% acetonitrile at 28 min. Optimum urea concentration was found to be 0.5 M and 24 h reaction time was sufficient without any additive such as CaCl2 and Tween-20.

• KSBB Journal
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• v.18 no.4
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• pp.306-311
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• 2003

Solid-phase refolding of immobilized proteins can be an effective way to reuse an immobilized enzyme column. Oriented immobilization methods are known to provide higher activity of the immobilized enzymes. In this study, using recombinant EK (enterokinase) as a model enzyme and a fusion protein, that consisted of recombinant human growth hormone and six His tag that was linked by the peptide of EK-specific recognition sequence, as a model substrate, we evaluated two oriented immobilization methods, i. e., reductive alkylation of N-terminus ${\alpha}$-amine and affinity interaction between poly-histidine tag and Ni-NTA (nickel-nitrilotriacetic acid). The immobilization yield, activity and cleavage of the immobilized enzymes, and the yield of solid-phase refolding were compared. The Ni affinity immobilization and the covalent immobilization yields were about 100％ and 65％, respectively. But the specific activities were the same, about 50％ of that of the soluble enzyme. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. Covalently immobilized EK showed almost 100％ refolding yield but the affinity immobilized EK showed only 70％ yield, which suggested the covalent conjugation provided more rigid ‘reference structure’ for the solid-phase refolding. The monomeric hGH could be easily obtained by capturing the cleaved poly Histidine tag by the Ni affinity column.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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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.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.519-520
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• 2003

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.1
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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.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.513-516
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• 2003

고체상 풀림과 재접힘 공정을 통해서 EK의 활성이 회복되어지는 것을 확인할 수 있었다. 이는 고정화 효소를 사용함으로써 기존의 액상반응에서 불가능한 효소의 재사용 문제를 해결할 수 있다. 친화도보다는 다중 공유결합을 통한 효소의 고정화가 효소의 활성 회복에 높은 안정성을 가질 수 있다고 예상한다.

• Biomedical Science Letters
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• v.16 no.3
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• pp.201-206
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• 2010

In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

• KSBB Journal
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• v.21 no.3
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• pp.204-211
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• 2006

For the production of the recombinant human interferon-gamma(rhIFN-${\gamma}$) in Escherichia coli, human glucagon and ferritin heavy chain were used as fusion partners. Even though rhIFN-${\gamma}$ is expressed as an inclusion body form in E. coli because of strong hydrophobicity of itself, over 50% of fused rhIFN-${\gamma}$ was expressed as soluble form in E. coli $Origami^{TM}$(DE3) harboring pT7FH(HE)-IFN-${\gamma}$ which encodes ferritin heavy chain-fused rhIFN-${\gamma}$. In the case of using glucagon-ferritin heavy chain hybrid mutant as a fusion partner, 6X His-tag was additionally introduced to N-terminus of GFHM(HE)-IFN-${\gamma}$ for enhancing purification yields of rhIFN-${\gamma}$. Fusion protein HGFHM(HE)-IFN-${\gamma}$ with two 6X His-tag was more effectively bound to Ni-NTA agarose bead than GFHM(HE)-IFN-${\gamma}$ with a 6X His-tag. rhIFN-${\gamma}$ was completely purified from enterokinase-treated HGFHM(HE)-IFN-${\gamma}$ by Ni-NTA affinity column. For high-level production of rhIFN-${\gamma}$, glucose was used as the sole carbon source with simple exponential feeding rate($2.4{\sim}7.2g/h$) in fed-batch process. The effective lactose concentration for the expression of the rhIFN-${\gamma}$ was $10{\sim}20mM$. Under the fed-batch culture conditions, rhIFN-${\gamma}$ production yield reached 11 g DCW/L for 6 hours after lactose induction.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.555-559
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• 2003

EK를 고정화하기 위해 니켈 친화결합 방법과 공유 결합형 고정화 방법을 수행하였으며 니켈 친화결합이 공유 결합형 고정화보다 높은 고정화 수율과 activity를 나타냈다. 풀림과 재접힘을 이용한 효소의 활성 회복은 공유결합형 고정화가 니켈 친화결합보다 높은 결과를 나타내었다. 또한 기질의 분자량 크기에 따른 절단율의 차이가 없었으므로 레진 공극 내부로의 확산도 차이에 의한 절단반응의 차이는 없는 것으로 나타났고, 기질 종류에 따른 EK의 활성은 작은 기질이 큰 기질보다 높은 활성을 보였다.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.4
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• pp.576-584
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• 2017

Objective: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ${\beta}-defensin-2$ (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. Methods: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. Results: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and grampositive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. Conclusion: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.