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Research Support, Non-U.S. Gov't
- Low-Scale Expression and Purification of an Active Putative Iduronate 2-Sulfate Sulfatase-Like Enzyme from Escherichia coli K12
- Edwin David Morales-Álvarez , Claudia Marcela Rivera-Hoyos , Angélica María Baena-Moncada , Patricia Landázuri , Raúl A. Poutou-Piñales , Homero Sáenz-Suárez , Luis A. Barrera , Olga Y. Echeverri-Peña
- J. Microbiol. 2013;51(2):213-221. Published online April 27, 2013
- DOI: https://doi.org/10.1007/s12275-013-2416-2
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- 15 Citations
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Abstract
- The sulfatase family involves a group of enzymes with a large degree of similarity. Until now, sixteen human sulfatases have been identified, most of them found in lysosomes. Human deficiency of sulfatases generates various genetic disorders characterized by abnormal accumulation of sulfated intermediate compounds. Mucopolysaccharidosis type II is characterized by the deficiency of iduronate 2-sulfate sulfatase (IDS), causing the lysosomal accumulation of heparan and dermatan sulfates. Currently, there are several cases of genetic diseases treated with enzyme replacement therapy, which have generated a great interest in the development of systems for recombinant protein expression. In this work we expressed the human recombinant IDS-Like enzyme (hrIDS-Like) in Escherichia coli DH5α. The enzyme concentration revealed by ELISA varied from 78.13 to 94.35 ng/ml and the specific activity varied from 34.20 to 25.97 nmol/h/mg. Western blotting done after affinity chromatography purification showed a single band of approximately 40 kDa, which was recognized by an IgY polyclonal antibody that was developed against the specific peptide of the native protein. Our 100 ml-shake-flask assays allowed us to improve the enzyme activity seven fold, compared to the E. coli JM109/pUC13-hrIDS-Like system. Additionally, the results obtained in the present study were equal to those obtained with the Pichia pastoris GS1115/ pPIC-9-hrIDS-Like system (3 L bioreactor scale). The system used in this work (E. coli DH5α/pGEX-3X-hrIDS-Like) emerges as a strategy for improving protein expression and purification, aimed at recombinant protein chemical characterization, future laboratory assays for enzyme replacement therapy, and as new evidence of active putative sulfatase production in E. coli.
Journal Articles
- Effects of Nicotine on the Growth and Protein Expression of Porphyromonas gingivalis
- Orson Baek , Weidong Zhu , Hyeong C. Kim Kim , Seok-Woo Lee Lee
- J. Microbiol. 2012;50(1):143-148. Published online February 27, 2012
- DOI: https://doi.org/10.1007/s12275-012-1212-8
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- 16 Citations
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Abstract
- Tobacco smoking is considered one of the most significant environmental risk factors for destructive periodontal disease. The effect of smoking on periodontopathic microbiota has not yet been elucidated, as previous studies failed to identify a concrete relationship between periodontopathic microorganisms and smoking. However, it is likely that smoking, as an environmental stress factor, may affect the behavior of dental plaque microorganisms, ultimately leading to alteration of the host-parasite interaction. The goal of this study was to examine the effect of nicotine, a major component of tobacco, on the growth and protein expression of the crucial periodontal pathogen Porphyromonas gingivalis. The growth of P. gingivalis 381 was measured after bacterial cells were cultivated in liquid broth containing various nicotine concentrations. First, P. gingivalis cells were allowed to grow in the presence of a single dose of nicotine (the single exposure protocol) at 0, 1, 2, 4, and 8 mg/L, respectively. Second, P. gingivalis cells were exposed to five consecutive doses of nicotine (the multiple exposure protocol) at 0, 1, 2, and 4 mg/L, respectively. Bacterial growth was measured by optical density and protein expression was analyzed by SDS-PAGE and 2-D gel electrophoresis. In the single nicotine exposure protocol, it was observed that the growth of P. gingivalis 381 was inhibited by nicotine in a dose-dependent manner. In the multiple nicotine exposure protocol, the growth rate of P. gingivalis increased with each subsequent nicotine exposure, even though bacterial growth was also inhibited in a dose dependent fashion. SDS-PAGE and 2-D gel electrophoresis analyses revealed a minor change in the pattern of protein expression, showing differences in proteins with low molecular weights (around 20 kDa) on exposure to nicotine. The results of this study suggest that nicotine exerts an inhibitory effect on the growth of P. gingivalis, and has a potential to modulate protein expression in P. gingivalis.
- Biologically Active and C-Amidated HinnavinII-38-Asn Produced from a Trx Fusion Construct in Escherichia coli
- Chang Soo Kang , Seung-Yeol Son , In Seok Bang
- J. Microbiol. 2008;46(6):656-661. Published online December 24, 2008
- DOI: https://doi.org/10.1007/s12275-008-0214-z
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- 9 Citations
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Abstract
- The cabbage butterfly (Artogeia rapae) antimicrobial peptide hinnavinII as a member of cecropin family is synthesized as 37 residues in size with an amidated lysine at C-terminus and shows the humoral immune response to a bacterial invasion. In this work, a synthetic gene for hinnavinII-38-Asn (HIN) with an additional amino acid asparagine residue containing amide group at C-terminus was cloned into pET-32a(+) vector to allow expression of HIN as a Trx fusion protein in Escherichia coli strain BL21 (DE3) pLysS. The resulting expression level of the fusion protein Trx-HIN could reach 15~20% of the total cell proteins and more than 70% of the target proteins were in soluble form. The fusion protein could be purified successfully by HiTrap Chelating HP column and a high yield of 15 mg purified fusion protein was obtained from 80 ml E. coli culture. Recombinant HIN was readily obtained by enterokinase cleavage of the fusion protein followed by FPLC chromatography, and 3.18 mg pure active recombinant HIN was obtained from 80 ml culture. The molecular mass of recombinant HIN determined by MALDI-TOF mass spectrometer is 4252.084 Da which matches the theoretical mass (4252.0 Da) of HIN. Comparing the antimicrobial activities of the recombinant hinnavinII with C-amidated terminus to that without an amidated C-terminus, we found that the amide of asparagine at C-terminus of hinnavinII improved its potency on certain microorganism such as E. coli, Enterobacter cloacae, Bacillus megaterium, and Staphylococcus aureus.
Research Support, Non-U.S. Gov't
- Protein Expression Analysis of Halobacillus dabanensis D-8T Subjected to Salt Shock
- De Qin Feng , Bo Zhang , Wei Dong Lu , Su Sheng Yang
- J. Microbiol. 2006;44(4):369-374.
- DOI: https://doi.org/2418 [pii]
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Abstract
- To investigate the mechanism of salt tolerance of gram-positive moderately halophilic bacteria, two-dimensional gel electrophoresis (2-D PAGE) was employed to achieve high resolution maps of proteins of Halobacillus dabanensis D-8T. Approximately 700 spots of proteins were identified from these 2-D PAGE maps. The majority of these proteins had molecular weights between 17.5 and 66 kDa, and most of them were distributed between the isoelectric points (pI) 4.0 and 5.9. Some protein spots were distributed in the more acidic region of the 2-D gel (pI <4.0). This pattern indicated that a number of proteins in the strain D-8T are acidic. To understand the adaptation mechanisms of moderately halophilic bacteria in response to sudden environmental changes, differential protein profiles of this strain were investigated by 2-D PAGE and ImagemasterTM 2D Platinum software after the cells were subjected to salt shock of 1 to 25% salinity for 5 and 50 min. Analysis showed 59 proteins with an altered level of expression as the result of the exposure to salt shock. Eighteen proteins had increased expression, 8 proteins were induced, and the expression of 33 proteins was down-regulated. Eight of the up-regulated proteins were identified using MALDI-TOF/MS and MASCOT, and were similar to proteins involved in signal transduction, proteins participating in energy metabolism pathways and proteins involved in stress.
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