• Journal of Microbiology and Biotechnology
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• v.33 no.11
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• pp.1513-1520
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• 2023

Kex2 protease (Kex2p) is a membrane-bound serine protease responsible for the proteolytic maturation of various secretory proteins by cleaving after dibasic residues in the late Golgi network. In this study, we present an application of Kex2p as an alternative endoprotease for the in vitro processing of recombinant fusion proteins produced by the yeast Saccharomyces cerevisiae. The proteins were expressed with a fusion partner connected by a Kex2p cleavage sequence for enhanced expression and easy purification. To avoid in vivo processing of fusion proteins by Kex2p during secretion and to guarantee efficient removal of the fusion partners by in vitro Kex2p processing, P₁', P₂', P₄, and P₃ sites of Kex2p cleavage sites were elaborately manipulated. The general use of Kex2p in recombinant protein production was confirmed using several recombinant proteins.

• 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.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.593-598
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• 2005

A collagenolytic enzyme, produced by Vibrio vulnificus CYK279H, was purified by ultrafiltration, dialysis, Q-Sepharose ion exchange and Superdex-200 gel chromatography. The enzyme from the supernatant was purified 13.2 fold, with a yield of 11.4%. The molecular weight of the purified enzyme was estimated by SDS-PAGE to be approximately 35.0kDa. The N-terminal sequence of the enzyme was determined as Gly-Asp-Pro-Cys-Met-Pro-Ile-Ile-Ser-Asn. The optimum temperature and pH for the enzyme activity were $35^{\circ}C$ and 7.5, respectively. The enzyme activity was stable within the pH and temperature ranges 6.8-8.0 and $20{\sim}35^{\circ}C$, respectively. The purified enzyme was strongly activated by $Zn^{2+},\;Li^{2+},\;and\;Ca^{2+}$, but inhibited by $Cu^{2+}$. In addition, the enzyme was strongly inhibited by 1, 10-phenanthroline and EDTA. The purified enzyme was suggested to be a neutral metalloprotease.

• Proceedings of the KIEE Conference
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• 2008.07a
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• pp.1466-1467
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• 2008

We describe here a novel micro total analysis system for the purification and identification of the affinity-captured proteins. Also we demonstrated the mass analysis of the Carcinoembrionic antigen (CEA) and Alpha femtoprotein which were chosen as the target cancer marker. For MALDI-TOF analyses, the proteins should to be separated from a protein mixture and be concentrated when needed. This procedure usually takes a long time even before protease-digested samples are to be obtained from them. Here, we describe integrated and efficient micro chip for protein purification and digestion for MALDI-TOF analyses. At first, disease protein is purified by passing the micro chamber from a protein mixture or human whole serum and released from the micro affinity beads by thermal heating. Purified protein is then transfer to the hole for trypsin digestion. The final sample is analyzed by MALDI-TOF. All the processes could be finished successfully within one hour, which renders MALDI-TOF analyses of a target protein quite simple.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.29 no.6
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• pp.797-804
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• 1996

Two trypsins were purified from shrimp hepatopancreas through ammonium sulfate fractionation, Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Sephacryl S-300 gel chromatography. Both enzymes had a single polypeptide chain with a molecular weight (M.W.) of 32 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis (SOS-PAGE), although trypsin A and B were estimated to be a molecular weight of 27.2 and 22.8 kDa, respectively, using Sephacryl S-300 gel filtration. Both trypsins had similar amino acid compositions and rich in glycine, valine, alanine, aspartic acid, and glutamic acid, but low in methionine and basic amino acids. Both enzymes were completely inactivated by soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine, leupeptin, however, not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) and pepstatin.

• Fisheries and Aquatic Sciences
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• v.1 no.2
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• pp.209-215
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• 1998

Trypsin-like enzyme was purified from shrimp hepatopancreas through Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, and Superdex 75 gel chromatography. Purity of trypsin-like enzyme was increased 69-fold with $44\%$ yield. The enzyme consisted of a single polypeptide chain with a molecular weight (M.W.) of 32 kDa judged by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was completely inactivated by serine enzyme inhibitors such as soybean trypsin inhibitor (SBTI), tosyl-L­lysine chloromethyl ketone (TLCK), and leupeptin. However, the enzyme was not affected by tosyl-L-phenylalanine chloromethyl ketone (TPCK) which is a chymotrypsin specific inhibitor. The enzyme had no activity against benzoyl-tyrosine ethyl ester (BTEE) which is a chymotrypsin specific substrate. The enzyme showed high activity on the carboxyl terminal of Phe, Tyr. Glu, Arg, and Asp. However. no activity was detected against the carboxyl terminal of Pro, Trp, Cys, Gly, Val, and Ala.

• Bulletin of the Korean Chemical Society
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• v.30 no.5
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• pp.1039-1042
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• 2009

RNA polymerase II C-terminal domain phosphatase 1 containing ubiquitin like domain (UBLCP1) has been identified as a regulatory molecule of RNA polymerase II. UBLCP1 consists of ubiquitin like domain (UBL) and phosphatase domain homologous with UDP and CTD phosphatase. UBLCP1 was cloned into the E.coli expression vectors, pET32a and pGEX 4T-1 with TEV protease cleavage site and purified using both affinity and gel-filtration chromatography. Domains of UBLCP1 protein were successfully purified as 7 mg/500 mL (UBLCP1, 36.78 KDa), 32 mg/500 mL (UBL, 9 KDa) and 8 mg/500 mL (phosphatase domain, 25 KDa) yielded in LB medium, respectively. Isotope-labeled samples including triple-labeled ($^2H/^{15}N/^{13}C$) UBLCP1 were also prepared for hetero-nuclear NMR experiments. $^{15}N-^{1}H$ 2D-HSQC spectra of UBLCP1 suggest that both UBL and phosphatase domain are properly folded and structurally independent each other. These data will promise us further structural investigation of UBLCP1 by NMR spectroscopy and/or X-ray crystallography.

• Biodesign
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• v.6 no.4
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• pp.96-99
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• 2018

The gram-positive bacterium Staphylococcus aureus is a common cause of abscesses, sinusitis and food poisoning. The emergence of antibiotic-resistant strains has caused significant clinical issues worldwide. The HslU-HslV complex was first identified as a prokaryotic homolog of eukaryotic proteasomes. HslU is an unfoldase that mediates the unfolding of the substrate proteins, and it works with the protease HslV in the complex. To date, the protein complex has been mostly studied in gram-negative bacteria. In this study, we report the purification and crystallization of the full-length HslU from S. aureus. The crystal diffracted X-rays to a $3.5{\AA}$ resolution, revealing that the crystals belong to space group $P2_1$, with unit cell parameters of a = 166.5, b = 189.6, $c=226.6{\AA}$, and ${\beta}=108.1^{\circ}$. We solved the phage problem by molecular replacement using the structure of HslU from Haemophilus influenzae as a search model. The cell content analysis with this molecular replacement solution revealed that 24 molecules are contained in the asymmetric unit. This structure provides insight into the structural and mechanistic difference of the HslUV complex of gram-positive bacteria.

• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• Journal of Life Science
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• v.19 no.5
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• pp.561-565
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• 2009

Human Tissue factor is an essential enzyme activator that forms a catalytic complex with factor VII/ VIIa, and catalyzes both the extrinsic and intrinsic blood coagulation cascades. The extracellular domain of human tissue factor is responsible for association with the biological partner. The efficient procedures for preparing biologically active human tissue factor are essential for the preclinical and clinical studies with coaguligands. An expression vector in Escherichia coli has been constructed to direct the production of extracellular human tissue factor without a fusion protein or a $His_6$ at the N-terminus. The recombinant human tissue factor was expressed in large amounts as a non-native state in E. coli. The recombinant protein was simply renatured during the DEAE-sephacel chromatographic purification procedure. Our expression and purification system does not require a protease treatment or an additional chromatographic step to remove a fusion contaminant, which provides a very useful alternative to conventional expression systems for the production of human tissue factor.