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Publication Open Access Functional characterization of two members of histidine phosphatase superfamily in Mycobacterium tuberculosis(2013) Olabisi Oluwabukola Coker; Saradee Warit; Kamolchanok Rukseree; Pijug Summpunn; Therdsak Prammananan; Prasit Palittapongarnpim; Mahidol University. Faculty of Science. Department of MicrobiologyBackground: Functional characterization of genes in important pathogenic bacteria such as Mycobacterium tuberculosis is imperative. Rv2135c, which was originally annotated as conserved hypothetical, has been found to be associated with membrane protein fractions of H37Rv strain. The gene appears to contain histidine phosphatase motif common to both cofactor-dependent phosphoglycerate mutases and acid phosphatases in the histidine phosphatase superfamily. The functions of many of the members of this superfamily are annotated based only on similarity to known proteins using automatic annotation systems, which can be erroneous. In addition, the motif at the N-terminal of Rv2135c is ‘RHA’ unlike ‘RHG’ found in most members of histidine phosphatase superfamily. These necessitate the need for its experimental characterization. The crystal structure of Rv0489, another member of the histidine phosphatase superfamily in M. tuberculosis, has been previously reported. However, its biochemical characteristics remain unknown. In this study, Rv2135c and Rv0489 from M. tuberculosis were cloned and expressed in Escherichia coli with 6 histidine residues tagged at the C terminal. Results: Characterization of the purified recombinant proteins revealed that Rv0489 possesses phosphoglycerate mutase activity while Rv2135c does not. However Rv2135c has an acid phosphatase activity with optimal pH of 5.8. Kinetic parameters of Rv2135c and Rv0489 are studied, confirming that Rv0489 is a cofactor dependent phosphoglycerate mutase of M. tuberculosis. Additional characterization showed that Rv2135c exists as a tetramer while Rv0489 as a dimer in solution. Conclusion: Most of the proteins orthologous to Rv2135c in other bacteria are annotated as phosphoglycerate mutases or hypothetical proteins. It is possible that they are actually phosphatases. Experimental characterization of a sufficiently large number of bacterial histidine phosphatases will increase the accuracy of the automatic annotation systems towards a better understanding of this important group of enzymes.Publication Open Access Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli.(2015-03) Usa Boonyuen; อุษา บุญยืน; Kamoltip Promnares; กมลทิพย์ พรหมณเรศ; Suwapat Junkree; Day, Nichloas P.J.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Mallika Imwong; Mahidol University. Faculty of Tropical Medicine. Mahidol Oxford Research Unit.; Mahidol University. Faculty of Tropical Medicine. Central Equipment Unit.; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.Human liver carboxylesterase 1 (CES1) plays a critical role in the hydrolysis of various ester- and amide-containing molecules, including active metabolites, drugs and prodrugs. However, it has been problematic to express recombinant CES1 in bacterial expression systems due to low solubility, with the CES1 protein being mainly expressed in inclusion bodies, accompanied by insufficient purity issues. In this study, we report an efficient in vitro method for refolding recombinant CES1 from inclusion bodies. A one-step purification with an immobilized-metal affinity column was utilized to purify His-tagged recombinant CES1. Conveniently, both denaturant and imidazole can be removed while the enzyme is refolded via buffer exchange, a dilution method. We show that the refolding of recombinant CES1 was successful in Tris-HCl at pH 7.5 containing a combination of 1% glycerol and 2mM β-mercaptoethanol, whereas a mixture of other additives (trehalose, sorbitol and sucrose) and β-mercaptoethanol failed to recover a functional protein. His-tagged recombinant CES1 retains its biological activity after refolding and can be used directly without removing the fusion tag. Altogether, our results provide an alternative method for obtaining a substantial amount of functionally active protein, which is advantageous for further investigations such as structural and functional studies.Publication Open Access Spatial distribution of potentially toxic trace elements of agricultural soils in the lower central plain of Thailand after the 2011 flood(2014-06) Aksarapak Pongpom; Kampanad Bhaktikul; Worachart Wisawapipat; Piyakarn Teartisup; Kampanad Bhaktikul; Mahidol University. Faculty of Environment and Natural Resource StudiesPublication Open Access Effective enhancement of Pseudomonas stutzeri D-phenylglycine aminotransferase functional expression in Pichia pastoris by co-expressing Escherichia coli GroEL-GroES(2012) Kanidtha Jariyachawalid; Poramaet Laowanapiban; Vithaya Meevootisom; Suthep Wiyakrutta; Mahidol University. Faculty of Science. Department of MicrobiologyBackground: D-phenylglycine aminotransferase (D-PhgAT) of Pseudomonas stutzeri ST-201 catalyzes the reversible stereo-inverting transamination potentially useful in the application for synthesis of D-phenylglycine and D-4-hydroxyphenylglycine using L-glutamate as a low cost amino donor substrate in one single step. The enzyme is a relatively hydrophobic homodimeric intracellular protein difficult to express in the soluble functionally active form. Over-expression of the dpgA gene in E. coli resulted in the majority of the D-PhgAT aggregated into insoluble inclusion bodies that failed to be re-natured. Expression in Pichia pastoris was explored as an alternative route for high level production of the D-PhgAT. Results: Intracellular expression of the codon-optimized synthetic dpgA gene under the PAOX1 promoter in P. pastoris resulted in inactive D-PhgAT associated with insoluble cellular fraction and very low level of D-PhgAT activity in the soluble fraction. Manipulation of culture conditions such as addition of sorbitol to induce intracellular accumulation of osmolytes, addition of benzyl alcohol to induce chaperone expression, or lowering incubation temperature to slow down protein expression and folding rates all failed to increase the active D-PhgAT yield. Co-expression of E. coli chaperonins GroEL-GroES with the D-PhgAT dramatically improved the soluble active enzyme production. Increasing gene dosage of both the dpgA and those of the chaperones further increased functional D-PhgAT yield up to 14400-fold higher than when the dpgA was expressed alone. Optimization of cultivation condition further increased D-PhgAT activity yield from the best co-expressing strain by 1.2-fold. Conclusions: This is the first report on the use of bacterial chaperones co-expressions to enhance functional intracellular expression of bacterial enzyme in P. pastoris. Only two bacterial chaperone genes groEL and groES were sufficient for dramatic enhancement of functionally active D-PhgAT expression in this yeast. With the optimized gene dosage and chaperone combinations, P. pastoris can be attractive for intracellular expression of bacterial proteins since it can grow to a very high cell density which is translated into the higher volumetric product yield than the E. coli or other bacterial systems.Publication Open Access Effect of cement film thickness on shear bond strengths of two resin cements.(2014-05) Somchai Urapepon; สมชาย อุรพีพล; Wutipong Luesak; Chatcharee Suchatlampong; ชัชรี สุชาติล้ําพงศ์; Somchai Urapepon; สมชาย อุรพีพล; Mahidol University. Faculty of Dentistry. Department of ProsthodonticsObjective: The purpose of this study was to determine the effect of cement thickness on the shear bond strength of two commercial adhesive resin cements, Panavia-F and Super-Bond C&B when used to bond nickel-chromium alloy to bovine dentine. Materials and methods: Ni-Cr alloy discs, Ø5x3 mm and bovine teeth (120 pairs) were bonded together by two different resin cements (Super Bond C&B and Panavia F) at four different film thicknesses (50, 100, 150 and 200 micrometers). After bonding, they were stored in 37°C water for 24 h. Shear bond test at a crosshead speed of 0.5mm/min was performed. The fracture surfaces of the specimens were observed by scanning electron microscope. Results: Panavia F showed the shear bond strength ranged from 4.02±1.44 MPa to 9.32±2.40 MPa, whereas Super Bond C&B showed a shear bond strength ranging from 5.01±1.38 MPa to 7.10±2.70 MPa. Both cements showed maximum shear bond strength to Ni-Cr alloy at 100 micrometers. In general, Panavia F showed higher bond strength than Super Bond C&B. Both cement thickness and cement type, and their interaction were found to have an effect on the shear bond strength of nickel-chromium alloy to bovine dentine. No significant differences were found when the film thickness was less than 150 micrometers for Panavia F (P>0.05) while those of Super Bond C&B were not significantly different at any film thickness (P>0.05). When the thickness of cement increased, more voids in the cement were found especially in Super Bond C&B. Conclusions: Within the limitations of this study, the shear bond strength between 50 and 150 micrometers was not significantly affected by the cement thickness.Publication Open Access การเปรียบเทียบความมีชีวิตของเซลล์ไฟโบรบลาสในน้ำยาแช่ฟันเดนท์ น้ำยาแฮงคส์บาลาสซอลท์ น้ำนม น้ำ และน้ำเกลือ(2014-01) Siriporn Timpawat; ศิริพร ทิมปาวัฒน์; Suwanna Korsuwannawong; สุวรรณา ก่อสุวรรณวงศ์; Ratchaporn Srichan; ราชพร สีจันทร์; Suwanna Korsuwannawong; สุวรรณา ก่อสุวรรณวงศ์; มหาวิทยาลัยมหิดล. คณะทันตแพทยศาสตร์. ภาควิชาทันตกรรมหัตถการและวิทยาเอ็นโดดอนต์; มหาวิทยาลัยมหิดล. คณะทันตแพทยศาสตร์. สำนักงานการวิจัย.35 น้ำนมให้ค่าร้อยละความมีชีวิตของเซลล์เท่ากับ 103.35±1.26 และ 116.56±2.23 น้ำให้ค่าร้อยละความชีวิตของเซลล์เท่ากับ 70.11±1.16 และ83.12±1.60 น้ำเกลือให้ค่าร้อยละความมีชีวิตของเซลล์เท่ากับ 101.95±1.76 และ 104.65±1.40 และเมื่อทดสอบที่ 90 นาที น้ำยาแช่ฟันเดนท์... 103.27±1.05% and 112.57±1.03%. Milk at room temperature and 4°C for 40 min were 103.35±1.26 % and 116.56 ±2.23% and at 90 min were 99.70±1.05% and 107.14±1.80%. Water at 37°C (Room Temperature) and 4°C for 40 min were 70.11±1.160% and 83.12±1.60