• Journal of Microbiology and Biotechnology
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• v.17 no.1
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• pp.29-36
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• 2007

The xynA gene encoding the xylanase A of Paenibacillus sp. DG-22 was isolated with a DNA probe obtained by PCR amplification, using degenerated primers deduced from the amino acid residues of the known N-terminal region of the purified enzyme and the conserved region in the family 11 xylanases. The positive clones were screened on the LB agar plates supplemented with xylan, by the Congo-red staining method. The xynA gene consists of a 630-bp open reading frame encoding a protein of 210 amino acids, and the XynA preprotein contains a 28-residues signal peptide whose cleavage yields a l82-residues mature protein of a calculated molecular weight of 20,000Da and pI value of 8.77. The cloned DNA fragment also has another ORF of 873 nucleotides that showed 76% identity to the putative transcriptional activator of Bacillus halodurans C-125. Most of the xylanase activity was found in the periplasmic space of E. coli. The xynA gene was subcloned into pQE60 expression vector to fuse with six histidine-tag. The recombinant xylanase A was purified by heating and immobilized metal affinity chromatography. The optimum pH and temperature of the purified enzyme were 6.0 and $60^{\circ}C$, respectively. This histidine-tagged xylanase A was less thermostable than the native enzyme.

• Journal of Microbiology and Biotechnology
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• v.25 no.2
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• pp.196-205
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• 2015

A Sulfolobus-E. coli shuttle vector for an efficient expression of the target gene in S. acidocaldarius strain was constructed. The plasmid-based vector pSM21 and its derivative pSM21N were generated based on the pUC18 and Sulfolobus cryptic plasmid pRN1. They carried the S. solfataricus P2 pyrEF gene for the selection marker, a multiple cloning site (MCS) with C-terminal histidine tag, and a constitutive promoter of the S. acidocaldarius gdhA gene for strong expression of the target gene, as well as the pBR322 origin and ampicillin-resistant gene for E. coli propagation. The advantage of pSM21 over other Sulfolobus shuttle vectors is that it contains a MCS and a histidine tag for the simple and easy cloning of a target gene as well as one-step purification by histidine affinity chromatography. For successful expression of the foreign genes, two genes from archaeal origins (PH0193 and Ta0298) were cloned into pSM21N and the functional expression was examined by enzyme activity assay. The recombinant PH0193 was successfully expressed under the control of the gdhA promoter and purified from the cultures by His-tag affinity chromatography. The yield was approximately 1 mg of protein per liter of cultures. The enzyme activity measurements of PH0913 and Ta0298 revealed that both proteins were expressed as an active form in S. acidocaldarius. These results indicate that the pSM21N shuttle vector can be used for the functional expression of foreign archaeal genes that form insoluble aggregates in the E. coli system.

• 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.

• The Journal of the Korean Society for Microbiology
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• v.35 no.5_6
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• pp.352-352
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• 2000

• Microbiology and Biotechnology Letters
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• v.46 no.3
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• pp.244-252
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• 2018

Protein disulfide isomerase A3 (PDIA3) is a major member of the protein disulfide isomerase (PDI) family. PDI proteins commonly reside in the endoplasmic reticulum and mediate important thiol-disulfide interchanges during post-translational protein folding. Unlike other PDI family members, PDIA3 is ubiquitous in various organ systems. However, its physiological activity varies in other tissues. PDIA3 has been associated with cancer, airway inflammation, neurodegenerative diseases, and metabolic diseases. However, the mechanisms of the association of PDIA3 with these pathological conditions remain unclear. Recombinant PDIA3 (rPDIA3) is needed to clarify the interactions between PDIA3 and certain physiological phenomena. In the present study, we aimed to produce highly purified rPDIA3 for use in pathological experiments. We expressed rPDIA3 with a histidine-enriched elongated peptide tag in Escherichia coli and obtained rPDIA3 at 97.8% purity using consecutive His-tag and reverse-phase chromatography. Elongated peptide tags screened from artificially designated library had dual functions for protein expression and simple purification.

• Biotechnology and Bioprocess Engineering:BBE
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• v.4 no.1
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• pp.72-77
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• 1999

An antibody containing a genetically engineered lipid group at the N-termunus and a hexahistidinyl tag at the C-terminus (Lpp-scF-His6) was immobilized in an oriented manner on the surface of liposome. Liposomes, consisting of antibody and phosphatidyl-choline, have been prepared and imaged by AFM. For AFM visualization, the resulting liposomes were bound on the surface of mica by two different mechanisms. The histidine tags present in the antibody molecules of the immonuliposome were anchored to the NiCl2 treated mica surface. Alternatively, the immunoliposomes were immunochemically bound on antigen-coated mica surface. Both approaches yielded liposomes which were clearly imaged without damage by AFM in ambient condition.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.539-542
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• 2003

Fusion gene of GFPuv and Cytochrome c-552 was inserted into the pTrcHis B vector and transferred to E. coli. A fusion protein of GFPuv and Cytochrome c-552 was expressed in BL21. This fusion protein was composed of a His-tag for purification using an immobilized metal affinity chromatography(IMAC). IMAC constitutes a rather facile means of unravelling the principles of recognition and, in particular, of identifying the counterligands on the protein surface, which interact with the ligated and immobilized metal ions. Histidine when present on the surface of a protein molecule under a favorable solvent condition, may serve as electron donors in coordination with the immobilized chelates of some transition metal ions$(Ni^{2+})$.

• Korean Journal of Microbiology
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• v.49 no.4
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• pp.309-312
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• 2013

Fungal cell walls consist of various glucans and chitin. Fungi produce chitinases for their growth and development. The inky cap, Coprinellus congregatus, produces at least two different chitinases during its life cycle. Chitinase 1 (chi1) is expresses throughout its life cycle while chitinase 2 (chi2) is expressed at the mushroom autolysing phase. The cloned cDNA of chi1 is successfully expressed as a fusion protein with c-myc in Pichia pastoris, and purified by the affinity chromatography. The optimum pH and temperature of Chi1 was pH 8.0 and $35^{\circ}C$, respectively when 4-nitrophenyl N,N',N"-triacetyl-${\beta}$-D-chitotrioside was used as the substrate. The $K_m$ value and $V_{max}$ for the substrate was 0.780 mM and 0.10 OD $min^{-1}unit^{-1}$, respectively. The addition of purified Chi1 resulted in total growth inhibition against several plant pathogenic fungi such as Alternaria alternata, Fusarium graminearum and Trichoderma harzianum at the concentration of 60 ${\mu}g/ml$.

• Journal of Applied Biological Chemistry
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• v.53 no.3
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• pp.132-138
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• 2010

A gene encoding a putative alanine racemase in B. pseudomycoides was cloned and expressed in Escherichia coli BL21(DE3) using a pET-21 vector harbouring 6xHistidine tag. Affinity purification of the recombinant alanine racemase with a nickel resin resulted in one band by SDS-PAGE analysis. The purified enzyme showed a molecular weight of 46 kDa. The enzyme was the most active toward L-alanine and secondly D-alanine, implying that the enzyme is an alanine racemase. D-cysteine significantly inhibited the enzyme activity and also L-cysteine to a lesser extent. The enzyme was considerably activated by addition of pyridoxal-5'-phosphate (PLP), showing that 73% increase in activity was observed at 0.3 mM, compared to control. The enzyme was the most active at pH 9.0 and more stable at alkaline pHs than acidic pHs.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.639-642
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• 2003

Olfactory receptor protein ODR10 was expressed in E.coli as fusion protein with GST and His6 Tag. Crude membrane extract of the expressed protein was coated on the surface of quartz crystal microbalance, and the interaction of the ODR10 with several odorants was examined. Although the expression level was very low, quartz crystal microbalance showed that the expressed protein interacted most strongly with diacetyl (butanedione), which is known to bind to the ODR10 protein selectively. The interaction between ODR10 and diacetyl was $5{\sim}10$ times stronger than the interaction between ODR10 and other odorants. Thus, E. coli cells expressing the olfactory receptor protein could be used as an olfactory biosensor. Also, such system could be used to test which olfactory receptor reacts specifically with which odorant molecules, since there has been no cheap and convenient way to test the interaction of olfactory receptors and odorant molecules yet.