• Applied Biological Chemistry
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• v.50 no.2
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• pp.107-110
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• 2007

The putative laccase gene, yacK of Escherichia coli, K-12 is not expressed in lab culture conditions. The laccase gene was amplified by PCR and subcloned into pET28a vector. The laccase overproduced in E. coli harboring pET28a was purified by His-affinity column chromatography. The purified laccase, which has the apparent molecular weight of 55,000 on the SDS-polyacrylamide gel showed enzyme activity on the guaiacol solution and agar plate. Optimum temperature and pH were around 65$^{\circ}C$ and 5.0, respectively.

• YAKHAK HOEJI
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• v.47 no.6
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• pp.410-416
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• 2003

Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High Five TM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C-termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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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.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.655-662
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• 1992

Pectate lyase produced by recombinant strain containing pectate lyase gene from alkalitolerant Bacillus sp. YA-14 was succesively purified with 257.6 purification folds and a 10.2% yields by the affinity method, eM-cellulose column chromatography followed by gel filtration on Sephadex G-I00 column. The optimal pH and temperature for pectate lyase activity were 10.0 and $60^{\circ}C$, respectively. The enzyme was stable between pH 4.0 and 10.0, and up to $50^{\circ}C$. The molecular weight of this enzyme was estimated to be 43,000 daltons by SDS-PAGE. Amino acid analysis showed that the enzyme contained more polar and basic amino acids, especially serine, glycine and tyrosine, than that of various pectate lyase from other strains. The N-terminal amino acid sequence was Ala-Asp-Leu-Gly-His-Gln-Thr.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.171-175
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• 2003

The putative endochitinase gene, yheB of Escherichia coli K-12 is not expressed under lab culture conditions. The endochitinase gene was amplified by PCR and subcloned into pET28c vector and pQE9 vector, respectively. The endochitinase produced in E. coli harboring pET28c containing yheB or pQE9 vector containing yheE was partly released into the growth medium. The overproduced endochitinase was partially purified by His affinity column chromatography and DE-52 column chromatography. The apparent molecular weight of the endochitinase determined by SDS-polyacrylamide gel electrophoresis was about 97,000. The purified E. coli endochitinase showed maximal chitinolytic activity at pH 6 and $40^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.784-788
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• 1995

The lambda Integrase (Int) carries out site-specific recombination between the two partner DNA sequences, attachment P (attP) and attachment B (attB). In order to study the recombination mechanism, a large quantity of pure integrase is required. Then, we constructed an int gene inserted recombinant plasmid (pNYL3) by using the pQE31 HIS-Tag vector, and produced the fusion protein, 6xHIS-Int from the E. coli TG1 strain carrying the pNYL3 plasmid. The recombinant protein produced was purified by phosphocellulose and Ni$^{++}$-NTA affinity column chromatographies. The result of the in vitro recombination assay using the standard reaction mixture containing 6xHIS-Int and partially purified integration host factor (IHF) showed that the 6xHIS-Int tagged recombination Integrase had the full recombination activity.

• BMB Reports
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• v.33 no.4
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• pp.294-299
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• 2000

Proteolytic processing of the dengue virus serotype 2 polyprotein precursor is catalyzed by a host signal peptidase and a virus encoded two-component protease consisting of the nonstructural proteins, NS2B and NS3. We expressed in Escherichia coli the NS2B, NS3(pro) and NS2B-NS3 proteins from the dengue virus type 2 strain 16681 as N-terminal fusions with a hexahistidine affinity tag under the control of the inducible trc promoter. All fusion proteins were purified to >90% purity by detergent extraction of inclusion bodies and a single step metal chelate chromatography. Proteins were refolded on-column and recovered with yields of 0.5, 6.0 and 1.0 mg/l of E. coli culture that was grown to $OD_{600}=1.0$ for NS2B, NS3(pro) and NS2B-NS3, respectively. Purified proteins gave strong signals in Western blots using $Ni^{2+}-nitrilotriacetic$ acid as a probe for the presence of the polyHis tag. During the purification process, $(His)_{6}NS2B-NS3$ was apparently not autoproteolytically cleaved at the NS2B/NS3 site.

• Journal of Microbiology and Biotechnology
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• v.19 no.7
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• BMB Reports
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• v.40 no.4
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• pp.604-609
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• 2007

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

• Proceedings of the Microbiological Society of Korea Conference
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• 2001.11a
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• pp.16-25
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• 2001

A genomic library of biphenyl-degrading strain Comamonas sp. SMN4 was constructed by using the cosmid vector pWE15 and introduced into Escherichia coli. Of 1,000 recombinant clones tested, two clones that expressed 2,3-dihydroxybiphenyl 1,2-dioxygenase activity were found (named pNB 1 and pNB2). From pNB1 clone, subclone pNA210, demonstrated 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, is isolated. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (23DBDO, BphC) is an extradiol-type dioxygenase that involved in third step of biphenyl degradation pathway. The nucleotide sequence of the Comamonas sp. SMN4 gene bphC, which encodes 23DBDO, was cloned into a plasmid pQE30. The His-tagged 23DBDO produced by a recombinant Escherichia coli, SG 13009 (pREP4)(pNPC), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 23DBDO construction was active. SDS-PAGE analysis of the purified active 23DBDO gave a single band of 32 kDa; this is in agreement with the size of the bphC coding region. The 23DBDO exhibited maximum activity at pH 9.0. The CD data for the pHs, showed that this enzyme had a typical a-helical folding structures at neutral pHs ranged from pH 4.5 to pH 9.0. This structure maintained up to pH 10.5. However, this high stable folding strucure was converted to unfolded structure in acidic region (pH 2.5) or in high pH (pH 12.0). The result of CD spectra observed with pH effects on 23DBDO activity, suggested that charge transition by pH change have affected change of conformational structure for 23DBDO catalytic reaction. The $K_m$ for 2,3-dihydroxybiphenyl, 3-metylcatechol, 4-methylcatechol and catechol was 11.7 $\mu$M, 24 $\mu$M, 50 mM and 625 $\mu$M.