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ABSTRACT: Ferritin-based nanoprobe (FBNP) hydrogel provides solutions to some of the traditional problems of bioassays. The schematic shows 3D and highly specific multiplex assays of markers for AIDS and Sjögren's syndrome using quantum-dot based reporters with different fluorescence emissions for the two syndromes. The advantages of using a hydrogel include that the stability of FBNPs is significantly enhanced within the hydrogel, FBNPs are distributed evenly throughout the hydrogel matrix, and the amount of FBNPs within the hydrogel can be well controlled.
Advanced Materials 07/2012; 24(35):4739-44, 4730. · 13.88 Impact Factor
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ABSTRACT: We synthesized fluorescent capsid nanoparticles (FCNPs) by genetically inserting fluorescent protein (FP) (DsRed or eGFP) into each of 240 surface spike tips of hepatitis B virus (HBV) capsid particles. That is, when expressed in E. coli, FCNPs formed spherical nanoparticles with uniform diameter of about 40 nm owing to the self-assembly function of HBV core protein (i.e. basic assembly unit of capsid) and were successfully purified through Ni(+2) affinity- and sucrose gradient based purification. We also added the glycine-rich fexible linker peptides in between DsRed (or eGFP) and capsid to reduce fluorescence quenching among the densely displayed DsReds (or eGFPs) on the capsid surface. As compared to cognate fluorescent monomer proteins, it is notable that FCNPs showed a significantly amplified (160-170-fold) fluorescence intensity and enhanced conformational stability even in 50% serum solutin at 37 °C. The high conformational stability of FCNPs seems to result both from the highly stable structure of HBV capsid particles and from the well oriented insertion of fluorescent protein into capsid spike tip to keep native conformation of DsRed or eGFP. When estimated with continuous exposure to strong excitation light, FCNPs also showed much higher photostability than DsRed, eGFP, and a commonly used organic fluorescent dye, which happened presumably because the enhanced conformational stability of FCNPs significantly reduced photobleaching of fluorophores. Especially, it is notable that rFCNPs stably emitted high-level fluorescence inside mouse for a prolonged period, thereby showing high in vivo stability. The developed FCNPs are likely to have a great potential to be used as an effective and non-cytotoxic tool for in vivo optical imaging as well as in vitro fluorescent reporter in various biomolecular detection assays.
Biomaterials 06/2012; 33(26):6194-200. · 7.40 Impact Factor
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ABSTRACT: Escherichia coli YrhB (10.6 kDa) from strain BL21(DE3) that is commonly used for protein overexpression is a stable chaperone-like protein and indispensable for supporting the growth of BL21(DE3) at 48 °C but not defined as conventional heat shock protein (HSP). YrhB effectively prevented heat-induced aggregation of ribonucleotide synthetase (PurK). Without ATP, YrhB alone promoted in vitro refolding of uridine phosphorylase (UDP) and protected thermal denaturation of the refolded UDP. As a cis-acting fusion partner, YrhB also significantly reduced inclusion body formation of nine aggregation-prone heterologous proteins in BL21(DE3). Unlike conventional small HSPs, YrhB remained monomer under heat shock condition.
FEBS letters 04/2012; 586(7):1044-8. · 3.54 Impact Factor
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ABSTRACT: As a fusion partner to express aggregation-prone heterologous proteins, we investigated the efficacy of Escherichia coli phosphoglycerate kinase (ePGK) that consists of two functional domains (N- and C-domain) and reportedly has a high structural stability. When the full-length ePGK (F-ePGK) was used as a fusion partner, the solubility of the heterologous proteins increased, but some of them still had a large fraction of insoluble aggregates. Surprisingly, the fusion expression using the N-domain of ePGK (N-ePGK) made the insoluble fraction significantly reduce to less than 10% for all the heterologous fusion proteins tested. Also, we evaluated the efficacy of N-ePGK in making the target proteins be expressed with their own native function or structure. It was found that of human ferritin light chain, bacterial arginine deiminase, human granulocyte colony stimulating factor were synthesized evidently with the self-assembly function, L-arginine-degrading activity, and the correct secondary structure, respectively, through the fusion expression using N-ePGK. These results indicate that N-ePGK is a highly potent fusion partner that can be widely used for the synthesis of a variety of heterologous proteins in E. coli.
Biotechnology and Bioengineering 08/2011; 109(2):325-35. · 3.95 Impact Factor
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ABSTRACT: Through the proteome analysis of Escherichia coli BL21(DE3), we previously identified the stress-responsive protein, arsenate reductase (ArsC), that showed a high cytoplasmic solubility and a folding capacity even in the presence of stress-inducing reagents. In this study, we used ArsC as an N-terminal fusion partner to synthesize nine aggregation-prone proteins as water-soluble forms. As a result, solubility of the aggregation-prone proteins increased dramatically by the fusion of ArsC, due presumably to its tendency to facilitate the folding of target proteins. Also, we evaluated and confirmed the efficacy of ArsC-fusion expression in making the fusion-expressed target proteins have their own native function or structure. That is, the self-assembly function of human ferritin light chain, l-arginine-degrading function of arginine deiminase, and the correct secondary structure of human granulocyte colony stimulating factor were clearly observed through transmission electron microscope analysis, colorimetric enzyme activity assay, and circular dichroism, respectively. It is strongly suggested that ArsC can be in general an efficient fusion expression partner for the production of soluble and active heterologous proteins in E. coli.
Enzyme and microbial technology. 07/2011; 49(2):124-30.
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ABSTRACT: We synthesized fluorescent ferritin nanoparticles (FFNPs) through bacterial expression of the hybrid gene consisting of human ferritin heavy chain (hFTN-H), spacer (glycine-rich peptide), and enhanced green (or red) fluorescent protein [eGFP (or DsRed)] genes. The self-assembly activity of hFTN-H that leads to the formation of nanoparticles (12 nm in diameter), the conformational flexibility of the C-terminus of hFTN-H, and the glycine-rich spacer enabled eGFPs (or DsReds) to be well displayed on the surface of each ferritin nanoparticle, resulting in the construction of green (or red) FFNPs [gFFNPs (or rFFNPs)]. As compared to eGFP (or DsRed) alone, it is notable that the developed FFNPs showed significantly amplified fluorescence intensity and also enhanced stability. DNA aptamers were chemically conjugated to gFFNP via each eGFP's cysteine residue that was newly introduced through site-directed mutagenesis (Ser175Cys). The DNA-aptamer-conjugated gFFNPs were used as a fluorescent reporter probe in the aptamer-based "sandwich" assay of a cancer marker [i.e., platelet-derived growth factor B-chain homodimer (PDGF-BB)] in phosphate-buffered saline buffer or diluted human serum. This is a simple two-step assay without any additional steps for signal amplification, showing that compared to the same aptamer-based assays using eGFP alone or Cy3, the detection signals, affinity of the reporter probe to the cancer marker, and assay sensitivity were significantly enhanced; i.e., the limit of detection was lowered to the 100 fM level. Although the PDGF-BB assay is reported here as a proof-of-concept, the developed FFNPs can be applied in general to any aptamer-based sandwich assays.
Analytical Chemistry 06/2011; 83(15):5834-43. · 5.86 Impact Factor
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ABSTRACT: Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4°C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10weeks at various storage temperatures (-20°C, 4°C, 25°C, and 37°C). During the period of 10weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.
Biochemical and Biophysical Research Communications 03/2011; 408(2):225-9. · 2.48 Impact Factor
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Advanced Functional Materials 09/2010; 20(23):4055 - 4061. · 10.18 Impact Factor
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Advanced Functional Materials 05/2010; 20(12):2004 - 2009. · 10.18 Impact Factor
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ABSTRACT: We previously reported that under the stress condition caused by the addition of 2-hydroxyethyl disulfide, a thiol-specific oxidant, to growing cultures of Escherichia coli BL21(DE3), a population of stress-responsive proteins [peptidyl-prolyl cis-trans isomerase B (PpiB), bacterioferritin (Bfr), putative HTH-type transcriptional regulator yjdC (YjdC), dihydrofolate reductase (FolA), chemotaxis protein cheZ (CheZ), and glutathione synthetase (GshB)] were significantly upregulated when compared with the nonstress condition. When those stress-responsive proteins were used as fusion partners for the expression of human granulocyte colony-stimulating factor (hG-CSF), the solubility of hG-CSF was dramatically enhanced in E. coli cytoplasm, whereas almost all of the directly expressed hG-CSF were aggregated to inclusion bodies. In addition, the spectra of circular dichroism measured with the purified hG-CSF were identical to that of standard hG-CSF, implying that the synthesized hG-CSF has native conformation. These results indicate that the bacterial stress-responsive proteins could be potent fusion expression partners for aggregation-prone heterologous proteins in E. coli cytoplasm.
FEMS Microbiology Letters 06/2009; 296(1):60-6. · 2.04 Impact Factor
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ABSTRACT: Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction1, 2, 3, 4, 5 can reduce the risk of death from heart attacks6, 7, 8, 9, 10. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range11. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays11, 12, 13, 14, 15, 16. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.
Nature Nanotechnology 03/2009; 4(4):259-264. · 27.27 Impact Factor
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ABSTRACT: Candida antarctica lipase B (CalB) was functionally expressed in the cytoplasm of Escherichia coli Origami(DE3) with the N-terminus fusion of E. coli endogenous proteins. The previously-identified stress responsive proteins through comparative proteome analyses such as malate dehydrogenase (Mdh), spermidine/putrescine-binding periplasmic protein (PotD), and FKBP-type peptidyl-prolyl cis-trans isomerase (PPIases) (SlyD) dramatically increased the solubility of CalB in E. coli cytoplasm when used as N-terminus fusion partners. We demonstrated that Mdh, PotD, and SlyD were powerful solubility enhancers that presumably facilitated the protein folding of CalB. Moreover, among the various fusion mutants, Mdh-CalB showed the highest hydrolytic activity and was as biologically active as standard CalB. Similarly to the previous report, the electrophoretic properties of CalB indicate that CalB seems to form dimer-based oligomer structures. We evaluated the structural compatibility between the fusion partner protein and CalB, which seems to be of crucial importance upon the bioactive dimer formation of CalB and might affect the substrate accessibility to the enzyme active site, thereby determining the biological activities of the fusion mutants.
Biochimica et Biophysica Acta 01/2009; 1794(3):519-25. · 4.66 Impact Factor
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ABSTRACT: The biochemical and physical properties of hepatitis B virus (HBV) small surface antigen (S-HBVsAg) from Berna Biotech Korea Corp. were systematically analyzed and characterized. Through various electrophoresis and immunoblotting assay of S-HBVsAg and its proteolytic products, it was confirmed that the S-HBVsAg vaccine particles are present in the form of covalent multimers that are assembled via strong intermolecular disulfide bonds. The S-HBVsAg particles contain no N-glycosylation moiety but some O-glycosidically linked mannoses. Evidently from N-terminus sequencing of both monomers and dimers that are formed by complete and partial reduction, respectively, of the S-HBVsAg particles under reducing SDS-PAGE condition, it is evident that each polypeptide within S-HBVsAg particles has authentic sequence of N-terminus. Denaturation plot shows that the S-HBVsAg vaccine particles were extremely stable especially in the solution with high acidity. This stability property of S-HBVsAg vaccine particles could provide very useful information for the optimization of the downstream process of recombinant S-HBVsAg particles synthesized from yeast cultures.
Vaccine 09/2008; 26(33):4138-44. · 3.77 Impact Factor
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ABSTRACT: Through 2-DE based quantitative proteomics, the dynamic characteristics of overall proteome profiles of Escherichia coli BL21(DE3) were systematically analyzed in the presence of four different stressors. Dithiothreitol and 2-hydroxyethyl disulfide are a reducing and an oxidizing agent, respectively, which disturb the redox balance in cytoplasm, while guanidine hydrochloride and heat shock are protein denaturants influencing protein folding. Heat shock proteins/foldases, transcription/translation-related proteins, various metabolic enzymes, and other stress regulatory proteins were found to be significantly up-regulated in response to the stressors. Heat shock proteins and translation-related proteins were generally responsive to almost all stress conditions. Two stressors, oxidative stress and guanidine hydrochloride-derived protein denaturation commonly induced the up-regulation of proteins related to transcription, whereas metabolic enzymes showed stress responses especially to the treatment of guanidine hydrochloride and heat shock. Similarities and differences of stress responses and protein-protein interactions of 80 proteins were systematically compared, and of special note, proteome-based stress-responsive proteins identified in the present study included 26 proteins that are being reported for the first time. The quantitative and systematic proteome analyses that we have performed provide more detailed information on E. coli BL21(DE3), a widely used host strain for recombinant protein overexpression.
Journal of Proteome Research 06/2008; 7(5):1891-903. · 5.11 Impact Factor
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ABSTRACT: The most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. Although commercial fusion partners including maltose binding protein and glutathione-S-transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. Thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study.
We analyzed the Escherichia coli proteome response to the exogenous stress of guanidine hydrochloride using 2-dimensional gel electrophoresis and found that RpoS (RNA polymerase sigma factor) was significantly stress responsive. While under the stress condition the total number of soluble proteins decreased by about 7 %, but a 6-fold increase in the level of RpoS was observed, indicating that RpoS is a stress-induced protein. As an N-terminus fusion expression partner, RpoS increased significantly the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm, indicating that RpoS is a very effective solubility enhancer for the synthesis of many recombinant proteins. RpoS was also well suited for the production of a biologically active fusion mutant of Pseudomonas putida cutinase.
RpoS is highly effective as a strong solubility enhancer for aggregation-prone heterologous proteins when it is used as a fusion expression partner in an E. coli expression system. The results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase.
BMC Biotechnology 02/2008; 8:15. · 2.35 Impact Factor
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ABSTRACT: The Escherichia coli proteome response to the stressor GdnHCl was analyzed through 2-dimensional gel electrophoresis (2-DE). We identified PotD (spermidine/putrescine-binding periplasmic protein) and Crr [glucose-specific phosphotransferase (PTS) enzyme IIA component] as a stress-responsive protein. Even under a stress situation where the total number of soluble proteins decreased by about 10%, 3.5- and 2.2-fold increase was observed in the synthesis of PotD and Crr, respectively. As fusion partners, PotD and Crr dramatically increased the solubility of many aggregation-prone heterologous proteins [e.g. human minipro-insulin (mp-INS), human epidermal growth factor (EGF), human prepro-ghrelin (ppGRN), human interleukin-2(hIL-2), human activation induced cytidine deaminase (AID), human glutamate decarboxylase (GAD(448-585)), Pseudomonas putida cutinase (CUT), human ferritin light chain (hFTN-L), human granulocyte colony-stimulating factor (G-CSF), and cold autoinflammatory syndrome1 protein (NALP3) Nacht domain (NACHT)] in the E. coli cytoplasm. Presumably PotD and Crr were very effective in shielding interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions leading to the formation of inclusion bodies most likely due to intrinsic high folding efficiency, chaperone-like activity, or a combination of both factors. Both the stress-induced proteins were well suited for the production of a biologically active fusion mutant of P. putida cutinase that can be expected to be of biotechnological and commercial interest.
Biochimica et Biophysica Acta 01/2008; 1774(12):1536-43. · 4.66 Impact Factor
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ABSTRACT: Abstract
Background
The most efficient method for enhancing solubility of recombinant proteins appears to use the fusion expression partners. Although commercial fusion partners including maltose binding protein and glutathione- S -transferase have shown good performance in enhancing the solubility, they cannot be used for the proprietory production of commercially value-added proteins and likely cannot serve as universal helpers to solve all protein solubility and folding issues. Thus, novel fusion partners will continue to be developed through systematic investigations including proteome mining presented in this study.
Results
We analyzed the Escherichia coli proteome response to the exogenous stress of guanidine hydrochloride using 2-dimensional gel electrophoresis and found that RpoS (RNA polymerase sigma factor) was significantly stress responsive. While under the stress condition the total number of soluble proteins decreased by about 7 %, but a 6-fold increase in the level of RpoS was observed, indicating that RpoS is a stress-induced protein. As an N-terminus fusion expression partner, RpoS increased significantly the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm, indicating that RpoS is a very effective solubility enhancer for the synthesis of many recombinant proteins. RpoS was also well suited for the production of a biologically active fusion mutant of Pseudomonas putida cutinase.
Conclusion
RpoS is highly effective as a strong solubility enhancer for aggregation-prone heterologous proteins when it is used as a fusion expression partner in an E. coli expression system. The results of these findings may, therefore, be useful in the production of other biologically active industrial enzymes, as successfully demonstrated by cutinase.
BMC Biotechnology. 01/2008;
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ABSTRACT: The proteome profile of Escherichia coli BL21(DE3) generated in response to heat shock stress was analyzed by two-dimensional electrophoresis (2-DE), wherein we identified a FKBP-type peptidyl-prolyl cis-trans isomerse (PPIases), SlyD, as a stress-responsive (i.e. aggregation-resistant) protein. Even under an imposed severe stress condition where 29 out of 858 soluble proteins were totally eliminated and the synthesis levels of 171 proteins decreased over 5-fold, a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition. As a fusion partner, as well as solubility enhancer, SlyD facilitated folding and significantly increased the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm. SlyD was very effective in sequestering interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions and the formation of inclusion bodies, most likely as a result of intrinsic folding efficiencies and/or chaperone-like activities. SlyD was also shown to be suitable for the production of a biologically active fusion mutant of Pseudomonas putida cutinase that is of considerable biotechnological and commercial interest.
Protein Engineering Design and Selection 12/2007; 20(11):543-9. · 2.94 Impact Factor
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ABSTRACT: Using 2-dimensional gel electrophoresis, the Escherichia coli proteome response to a heat-shock stress was analyzed and a 1.6-fold increase of malate dehydrogenase was observed even under the heat-shock condition where the total number of soluble proteins decreased by about 5%. We subsequently demonstrated that, as an N-terminus fusion expression partner, malate dehydrogenase facilitated the folding of, and dramatically increased the solubility of, many aggregation-prone heterologous proteins in E. coli cytoplasm. Therefore, malate dehydrogenase is well suited for production of a biologically active fusion mutant of cutinase (Pseudomonas putida origin) that is currently of considerable to biotechnology and commercial industries.
Biotechnology Letters 11/2007; 29(10):1513-8. · 1.68 Impact Factor
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ABSTRACT: Through two-dimensional electrophoresis, Escherichia coli proteome response to a protein denaturant, guanidine hydrochloride, was analyzed and elongation factor Ts (Tsf) detected as a stress-induced protein. Many host proteins aggregated, or their synthesis levels decreased significantly under conditions of protein denaturation as 34 out of 699 soluble proteins knocked out and 63 proteins decreased by over 2.5-fold. Interestingly, the expression level of Tsf increased 1.61-fold compared with a nonstress condition. Contrary to direct expression, various heterologous proteins were solubly expressed in E. coli when subjected to N-terminus fusions of Tsf. Owing most likely to an intrinsic high folding efficiency, Tsf seemed to play critical roles in sequestering interactive surfaces of heterologous proteins from nonspecific protein-protein interactions leading to formation of inclusion bodies. It has been also demonstrated that Tsf is effective in aiding the production of a biologically active bacterial cutinase, which could be of interest to biotechnology and commercial applications.
FEMS Microbiology Letters 10/2007; 274(1):132-8. · 2.04 Impact Factor
