• Journal of Life Science
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• v.29 no.4
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• pp.492-498
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• 2019

Previously, three kinds of lipases, lipAD1, lipAD2, and lipAD3, and lipase chaperone, lipBD, of Acinetobacter schindleri DYL129 isolated from soil sample were reported. In this report, three lipase and lipase chaperone were cloned into the pET32a(+) or pGEX-6P-1 vectors for protein expression in Escherichia coli, and named as pETLAD1, pETLAD2, pETLAD3 and pETLBD or pGEXLAD1, pGEXLAD 2, pGEXLAD3 and pGEXLBD, respectively. Protein expression rate was higher in pET system than in pGEX system. Although LipAD1 and LipAD2 were produced as inclusion bodies, their expression levels were high. So LipAD1 and LipAD2 could be solubilized in 8 M urea buffer and purified. LipAD3 and LipBD were overexpressed in soluble form and purified. Those proteins were purified by His-tag affinity chromatography connected in AKTA prime system. The activities of the purified lipases were demonstrated with 1% tributyrin agar plate. After purification, molecular mass was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LipAD1 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl butyrate, LipAD2 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl myristate, and LipAD3 showed high activity toward ${\rho}$-nitrophenyl acetate, ${\rho}$-nitrophenyl butyrate, and ${\rho}$-nitrophenyl miristate, respectively. Three lipases, LipAD1, LipAD2, and LipAD3, showed optimal reaction at $50^{\circ}C$ using ${\rho}$-nitrophenyl butyrate, as substrate.

• Journal of Life Science
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• v.32 no.2
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• pp.135-141
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• 2022

Thymosin β-4 (TB4) is a small peptide composed of 43 amino acids. To obtain sufficient biologically active mouse TB4 economically, we cloned and overexpressed this gene in an Escherichia coli system. With the isopropyl β-D-1-thiogalactopyranoside induction of the E. coli transformant, TB4 fusion protein with intein- and chitin-binding domain was successfully expressed in the soluble fraction within the E. coli cell. The TB4-intein - chitin-binding domain fusion protein was purified from the soluble fraction of E. coli cell lysate. The affinity chromatography with chitin beads and dithiothreitol-mediated intein self-cleavage reaction releases the TB4 peptide into the stripping solution. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis and Western blot analyses were used to confirm that the recombinant TB4 peptide was produced with the expected size of 5 kDa. We found that the recombinant TB4 stimulated cell migration in the transwell plate chamber assay. After 18 hr of the treatment of the recombinant TB4 with 1 ng/ml concentration, the migration of the HT1080 cell was increased by 20% compared with that of the chemically synthesized TB4. The recombinant TB4 was also observed to promote the healing of a wound area in C57BL/6 mice by as high as 35% compared with that of the chemically synthesized TB4. These results suggest that the recombinant TB4 has better biological activity for cell migration and wound healing than that of the chemically synthesized TB4 peptide.

• Journal of Dental Rehabilitation and Applied Science
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• v.38 no.3
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• pp.150-161
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• 2022

Purpose: To determine the effects of the microgroove-fibronectin complex surface on the expression of various genes related to cellular activity in human gingival fibroblasts. Materials and Methods: Smooth titanium specimens (NE0), acid-treated titanium specimens (E0), microgroove and acid-treated titanium specimens (E60/10), fibronectin-fixed smooth titanium specimens (NE0FN), acid-treated and fibronectin-immobilized titanium specimens (E0FN), and microgroove and acid-treated titanium specimens immobilized with fibronectin (E60/10FN) were prepared. Real-time polymerase chain reaction experiments were conducted on 44 genes related to cell behavior of human gingival fibroblasts. Results: Adhesion and proliferation of human gingival fibroblast on microgroove-fibronectin complex titanium were activated through four types of signaling pathway. Integrin α5, Integrin β1, Integrin β3, Talin-2, which belong to the focal adhesion pathway, AKT1, AKT2, NF-κB, which belong to the PI3K-AKT signaling pathway, MEK2, ERK1, ERK2, which belong to the MAPK signaling pathway, and Cyclin D1, CDK4, CDK6 genes belonging to the cell cycle signaling pathway were upregulated on the microgroove-fibronectin complex titanium surface (E60/10FN). Conclusion: The microgroove-fibronectin complex titanium surface can up-regulate various genes involved in cell behavior.