xin quan - Academia.edu (original) (raw)
Papers by xin quan
Molecular cancer therapeutics, Jan 29, 2018
Cancer stem-like cells are hypothesized to be the major tumor initiating cell population of human... more Cancer stem-like cells are hypothesized to be the major tumor initiating cell population of human cutaneous squamous cell carcinoma (cSCC), but the landscape of molecular alterations underpinning their signaling and cellular phenotypes as drug targets remain undefined. In this study, we developed an experimental pipeline to isolate a highly enriched CD133+CD31-CD45-CD61-CD24- (CD133+) cell population from primary cSCC specimens by flow cytometry. The CD133+ cells show enhanced stem-like phenotypes, which were verified by spheroid and colony formation in vitro and tumor generation in vivo. Gene expression profiling of CD133+/- cells was compared and validated, and differentially expressed gene signatures and top pathways were identified. CD133+ cells expressed a repertoire of stemness and cancer related genes, including NOTCH and NOTCH1-mediated NF-kB pathway signaling. Other cancer-related genes from WNT, growth factor receptors, PI3K/mTOR, STAT pathways, and chromatin modifiers wer...
Journal of tissue engineering and regenerative medicine, Jan 4, 2016
Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regenerati... more Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regeneration. To address this issue, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin hydrogel was prepared and injected into Schwann cell (SC)-seeded collagen-chitosan conduits to increase oxygen supply to SCs within the conduits. The conduit containing PFTBA-SC gel was then applied to bridge a 15-mm sciatic nerve defect in rats. It was observed that most of the GFP-labeled SCs initially seeded in the PFTBA hydrogel remained alive for approximately 28 days after their in vivo implantation. The number of SCs was significantly higher in the PFTBA-SC scaffold than that in the SC scaffold without PFTBA. In addition, nerve regeneration and functional recovery were examined after nerve injury repair. We found that the PFTBA-SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons. Further studies showed the PFTBA-SC scaffold was able to accelerate t...
TELKOMNIKA Indonesian Journal of Electrical Engineering, 2014
In recent years, the growing power quality problems in smart grid cause widespread concern at hom... more In recent years, the growing power quality problems in smart grid cause widespread concern at home and abroad. Because the traditional power quality algorithms which are based on Nyquist sampling theory have the drawbacks of complicated, heavy computations and poor real-time performance when sampling and analyzing continuous massive signals in smart grid. This paper discussed an improved reconstruction algorithm based on compressed sensing due to the sparsity of power quality signals in frequency domain for power quality analysis. By using the ZigBee wireless gateway for wireless sensor networks and energy metering chip, we develop a single meter node to do relative experiments. In the condition of the real test-bed and several compared experiments, power quality information in the highly compression ratio has good performance according to CSR (Compression Sampling Ratio), SNR (Signal to Noise Ratio), MSE (Mean Squared Error) and ERP (Energy Recovery Percentage) , and will be widely used in power quality analysis.
Neoplasia, 2014
Cancer stem cells (CSC) and genes have been linked to cancer development and therapeutic resistan... more Cancer stem cells (CSC) and genes have been linked to cancer development and therapeutic resistance, but the signaling mechanisms regulating CSC genes and phenotype are incompletely understood. CK2 has emerged as a key signal serine/ threonine kinase that modulates diverse signal cascades regulating cell fate and growth. We previously showed that CK2 is often aberrantly expressed and activated in head and neck squamous cell carcinomas (HNSCC), concomitantly with mutant (mt) tumor suppressor TP53, and inactivation of its family member, TAp73. Unexpectedly, we observed that classical stem cell genes Nanog, Sox2, and Oct4, are overexpressed in HNSCC with inactivated TAp73 and mtTP53. However, the potential relationship between CK2, TAp73 inactivation, and CSC phenotype is unknown. We reveal that inhibition of CK2 by pharmacologic inhibitors or siRNA inhibits the expression of CSC genes and side population (SP), while enhancing TAp73 mRNA and protein expression. Conversely, CK2 inhibitor attenuation of CSC protein expression and the SP by was abrogated by TAp73 siRNA. Bioinformatic analysis uncovered a single predicted CK2 threonine phosphorylation site (T27) within the N-terminal transactivation domain of TAp73. Nuclear CK2 and TAp73 interaction, confirmed by co-immunoprecipitation, was attenuated by CK2 inhibitor, or a T27A point-mutation of this predicted CK2 threonine phospho-acceptor site of TAp73. Further, T27A mutation attenuated phosphorylation, while enhancing TAp73 function in repressing CSC gene expression and SP cells. A new CK2 inhibitor, CX-4945, inhibited CSC related SP cells, clonogenic survival, and spheroid formation. Our study unveils a novel regulatory mechanism whereby aberrant CK2 signaling inhibits TAp73 to promote the expression of CSC genes and phenotype.
Virus Research, 1996
The complete nucleotide sequence of the RNA 3 of hydrangea mosaic virus (HdMV) was determined. It... more The complete nucleotide sequence of the RNA 3 of hydrangea mosaic virus (HdMV) was determined. It consists of 2268 nucleotides and contains two open reading frames (ORF). ORF 1 encodes for a putative translation product of 293 amino acids which shared 64.9% identity with the 3a protein of tobacco streak virus (TSV). ORF 2 encodes for a putative translation product of 220 amino acids which shared 54.2% identity with the coat protein of TSV. The relationship between the proteins of HdMV and the corresponding proteins of ilarviruses other than TSV was more distant. No zinc-finger-like motif was found in the coat protein of HdMV but the N-terminus of the protein was rich in basic amino acids. Both terminal, non-coding regions of HdMV RNA 3 contained repeated sequences with corresponding homologous fragments in the RNA 3 of TSV. On the basis of the similarities between HdMV and TSV that we detected, we propose that HdMV be included in subgroup 1 of the genus Ilarvirus together with TSV.
Nature Biotechnology, 2010
Protein-Based Aqueous−Multiphasic Systems
Langmuir, 2010
This paper reports the formation of aqueous-multiphasic systems (AMPS) exclusively made using ela... more This paper reports the formation of aqueous-multiphasic systems (AMPS) exclusively made using elastin-like polypeptides (ELP) which have the ability to undergo reversible inverse phase transitions. Manipulating variables such as the salt concentration and the molecular weight and the composition of ELPs (using different amino acid sequences or by fusing the ELP with different functional proteins) permits modulation of the temperature at which phase transition takes place, the number of phases that are formed, and the composition of the multiple aqueous phases. Using these variables, isotropic hybrid colloids with tunable functionality (in this case, fluorescent intensity) and anisotropic colloids with variable morphologies could be generated. While formation of AMPS and anisotropic colloids has been reported in the literature using synthetic polymers, to our knowledge this is the first report of generating such systems using proteins.
Self-Cleavable Stimulus Responsive Tags for Protein Purification without Chromatography
Journal of the American Chemical Society, 2005
A simple method to purify recombinant proteins is described by fusing a target protein with an in... more A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.
In vivo and in vitro application of elastin-like polypeptides
Elastin-like polypeptides (ELP) are artificially designed protein biopolymers that can be produce... more Elastin-like polypeptides (ELP) are artificially designed protein biopolymers that can be produced by living organisms. These proteins have the unique ability to undergo reversible inverse phase transition, in response to changes in temperature and/or addition of chaotropic salts. Below the transition temperature (Tt), ELP is soluble in water. Increasing the temperature above Tt, ELP coacervates into an aqeous ELP-rich phase. In this thesis, this unique feature of ELP was used in for recombinant protein purification and for the formation of aqueous multiple-phase systems. ^ For protein purification, ELP was fused with an intein and a model protein (thioredoxin), to demonstrate a simple and inexpensive approach for recombinant protein purification. The ELP tags replace the chromatographic media and the intein replaces the use of the protease in conventional methods. Using ELP tags was found to be consistent with large-scale recombinant protein production/purification by purifying an ...
The complete nucleotide sequence of RNA 3 of citrus leaf rugose and citrus variegation ilarviruses
Journal of General Virology, 1995
The complete nucleotide sequence of the RNA 3 of lilac ring mottle ilarvirus
Journal of General Virology, 1995
In vivo formation of protein based aqueous microcompartments
Journal of the American Chemical Society, 2009
In this paper, we report the formation of protein based liquid droplets resulting in the formatio... more In this paper, we report the formation of protein based liquid droplets resulting in the formation of in vivo microcompartments in E. coli or tobacco cells. These microcompartments were generated by expressing elastin-like polypeptides (ELP), which have the ability to undergo a reversible phase transition, resulting in the formation of an aqueous two-phase system (ATPS) in the cytoplasm of the cell. We prove that these microcompartments are liquid by expressing a fusion protein consisting of ELP and GFP and by performing fluorescence recovery after photobleaching (FRAP) experiments at different stages of cell cultivation. In the initial phases of cell growth, the fusion protein concentration is low and is not sufficient to drive the formation of a second aqueous phase. As the intracellular fusion protein concentration increases with longer cultivation time, droplets start forming, and as protein expression continues, the droplets coalesce at the poles of the E. coli cells. FRAP expe...
Highly efficient synthesis of ordered mesoporous silica materials with controllable microporosity using surfactant mixtures as templates
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2006
Ordered mesoporous silica materials (SBA-family) with 2-D hexagonal (p6mm) and 3-D cubic (Im3m) s... more Ordered mesoporous silica materials (SBA-family) with 2-D hexagonal (p6mm) and 3-D cubic (Im3m) symmetry have been efficiently synthesized by using the mixture of triblock copolymer surfactant and sodium dodecylsulfonate as co-templates at relatively low ...
Chinese Science Bulletin, 2011
Uropathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary trac... more Uropathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary tract infection (UTI). Several UPEC virulence factors have been identified, but more are yet to be found. We previously identified a novel 789-bp-long DNA fragment (named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique. In the present study, we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity. Through bioinformatic analysis, we predicted that this gene is a 1311-bp open reading frame (ORF), which we designated ORF R049 (GenBank accession No.: EF488001). We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography. Using mouse immunosera generated by the purified protein, we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting. To test the potential of this protein as a vaccine candidate, we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract. The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group. However, the degree of renal pathological damage was not significantly improved in the immunized mice. Our study has identified a novel gene of UPEC which can generate protective immunity against UTI. This novel gene provides a promising new vaccine candidate. uropathogenic Escherichia coli, genome walking, cloning and expression, immunoprotection
Purification of an elastin-like fusion protein by microfiltration
Biotechnology and Bioengineering, 2006
This article describes a simple and potentially scalable microfiltration method for purification ... more This article describes a simple and potentially scalable microfiltration method for purification of recombinant proteins. This method is based on the fact that when an elastin-like polypeptide (ELP) is fused to a target protein, the inverse phase transition behavior of the ELP tag is imparted to the fusion protein. Triggering the phase transition of a solution of the ELP fusion protein by an increase in temperature, or isothermally by an increase in salt concentration, results in the formation of micron-sized aggregates of the ELP fusion protein. In this article, it is shown that these aggregates are efficiently retained by a microfiltration membrane, while contaminating E. coli proteins passed through the membrane upon washing. Upon reversing the phase transition by flow of Milli-Q water, soluble, pure, and functionally active protein is eluted from the membrane. Proof-of principle of this approach was demonstrated by purifying a fusion of thioredoxin with ELP (Trx-ELP) with greater than 95% recovery of protein and with greater than 95% purity (as estimated from SDS-PAGE gels). The simplicity of this method is demonstrated for laboratory scale purification by purifying Trx-ELP from cell lysate using a syringe and a disposable microfiltration cartridge. The potential scalability of this purification as an automated, continuous industrial-scale process is also demonstrated using a continuous stirred cell equipped with a microfiltration membrane.
Rapid construction and characterization of synthetic antibody libraries without DNA amplification
Biotechnology and Bioengineering, 2010
We report on a simple method to rapidly generate very large libraries of genes encoding mutant pr... more We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.
Biomacromolecules, 2006
In this paper, we demonstrate proof-of-principle for a method that allows selective recovery of m... more In this paper, we demonstrate proof-of-principle for a method that allows selective recovery of molecules present at very low concentrations in complex mixtures. The method makes use of an elastin-like polypeptide (ELP) as a coaggregant for the capture of an ELP tagged recombinant protein present at concentrations as low as 10 pM, with a recovery higher than 90%. This coaggregation process was found to be independent of the concentration, at least up to 10 pM concentration of the ELP tagged protein. The coaggregation process is highly specific as was demonstrated by spiking crude cell lysate with the ELP tagged recombinant protein to a final concentration of 1 nM and recovering more than 80% of it to a high level of purity. The method should be particularly useful for high-throughput proteomic studies, where small amounts of poorly expressed proteins could be recovered for analysis by mass spectrometry. In a more general context, the concept presented in this paper provides a method that is highly efficient, specific, and fully reversible, which should render it useful in areas other than recombinant protein purification.
Archives of Virology, 2003
ACS Applied Materials & Interfaces, 2014
Morphological evolution associated with silicidation of Co thin films deposited on (100) and (1 1... more Morphological evolution associated with silicidation of Co thin films deposited on (100) and (1 1 1) Si substrates has been followed using tra~lsmission electron microscopy with in situ thermal annealing from ambient temperature up to 850°C. Noticeable structural changes dssociated with the formation of CoSi, occur at temperatures as low as 400°C and the reaction is essentially complete at about 500°C. Prolonged heating above 500°C leads to CoSi, grain growth and coalescence and, finally, to pinholes formatio11. Silicidation of Co films on (100) and (1 11) Si substrates follo~vs the same pattern. The morphology of films annealed in situ is similar to those annealed ex situ except that the SilCoSi2 interface appears to be much rougher. This behavior is associated with the specific geometry of cross-sectional TEM specimens, where surface diffusion dominates bulk diffusion. Very thin Co films, which have less contribution from surface diffusion than thicker films, are ideal for studying dynamic phenomena at CoISi reactive interfaces.
XPS and AES investigation of nanometer composite coatings of NiPZnX on steel surface (ZnX = ZnSnO3, Zn3(PO4)2, ZnSiO3)
Applied Surface Science, 2001
In this paper, the physical chemistry nature of the NiPZnX coatings have been studied by weighi... more In this paper, the physical chemistry nature of the NiPZnX coatings have been studied by weighing method, accelerated corrosion tests, tarnish test, high-temperature oxidation tests, Auger electron spectroscopy (AES) and X-ray photoelectron spectroscopy (XPS). The result shows that the surfaces of the coatings are homogeneous, polished, solid and with strong corrosion resistance.
Molecular cancer therapeutics, Jan 29, 2018
Cancer stem-like cells are hypothesized to be the major tumor initiating cell population of human... more Cancer stem-like cells are hypothesized to be the major tumor initiating cell population of human cutaneous squamous cell carcinoma (cSCC), but the landscape of molecular alterations underpinning their signaling and cellular phenotypes as drug targets remain undefined. In this study, we developed an experimental pipeline to isolate a highly enriched CD133+CD31-CD45-CD61-CD24- (CD133+) cell population from primary cSCC specimens by flow cytometry. The CD133+ cells show enhanced stem-like phenotypes, which were verified by spheroid and colony formation in vitro and tumor generation in vivo. Gene expression profiling of CD133+/- cells was compared and validated, and differentially expressed gene signatures and top pathways were identified. CD133+ cells expressed a repertoire of stemness and cancer related genes, including NOTCH and NOTCH1-mediated NF-kB pathway signaling. Other cancer-related genes from WNT, growth factor receptors, PI3K/mTOR, STAT pathways, and chromatin modifiers wer...
Journal of tissue engineering and regenerative medicine, Jan 4, 2016
Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regenerati... more Local hypoxia in the early stages of peripheral nerve injury is a challenge for axonal regeneration. To address this issue, perfluorotributylamine (PFTBA)-based oxygen carrying fibrin hydrogel was prepared and injected into Schwann cell (SC)-seeded collagen-chitosan conduits to increase oxygen supply to SCs within the conduits. The conduit containing PFTBA-SC gel was then applied to bridge a 15-mm sciatic nerve defect in rats. It was observed that most of the GFP-labeled SCs initially seeded in the PFTBA hydrogel remained alive for approximately 28 days after their in vivo implantation. The number of SCs was significantly higher in the PFTBA-SC scaffold than that in the SC scaffold without PFTBA. In addition, nerve regeneration and functional recovery were examined after nerve injury repair. We found that the PFTBA-SC scaffold was capable of promoting axonal regeneration and remyelination of the regenerated axons. Further studies showed the PFTBA-SC scaffold was able to accelerate t...
TELKOMNIKA Indonesian Journal of Electrical Engineering, 2014
In recent years, the growing power quality problems in smart grid cause widespread concern at hom... more In recent years, the growing power quality problems in smart grid cause widespread concern at home and abroad. Because the traditional power quality algorithms which are based on Nyquist sampling theory have the drawbacks of complicated, heavy computations and poor real-time performance when sampling and analyzing continuous massive signals in smart grid. This paper discussed an improved reconstruction algorithm based on compressed sensing due to the sparsity of power quality signals in frequency domain for power quality analysis. By using the ZigBee wireless gateway for wireless sensor networks and energy metering chip, we develop a single meter node to do relative experiments. In the condition of the real test-bed and several compared experiments, power quality information in the highly compression ratio has good performance according to CSR (Compression Sampling Ratio), SNR (Signal to Noise Ratio), MSE (Mean Squared Error) and ERP (Energy Recovery Percentage) , and will be widely used in power quality analysis.
Neoplasia, 2014
Cancer stem cells (CSC) and genes have been linked to cancer development and therapeutic resistan... more Cancer stem cells (CSC) and genes have been linked to cancer development and therapeutic resistance, but the signaling mechanisms regulating CSC genes and phenotype are incompletely understood. CK2 has emerged as a key signal serine/ threonine kinase that modulates diverse signal cascades regulating cell fate and growth. We previously showed that CK2 is often aberrantly expressed and activated in head and neck squamous cell carcinomas (HNSCC), concomitantly with mutant (mt) tumor suppressor TP53, and inactivation of its family member, TAp73. Unexpectedly, we observed that classical stem cell genes Nanog, Sox2, and Oct4, are overexpressed in HNSCC with inactivated TAp73 and mtTP53. However, the potential relationship between CK2, TAp73 inactivation, and CSC phenotype is unknown. We reveal that inhibition of CK2 by pharmacologic inhibitors or siRNA inhibits the expression of CSC genes and side population (SP), while enhancing TAp73 mRNA and protein expression. Conversely, CK2 inhibitor attenuation of CSC protein expression and the SP by was abrogated by TAp73 siRNA. Bioinformatic analysis uncovered a single predicted CK2 threonine phosphorylation site (T27) within the N-terminal transactivation domain of TAp73. Nuclear CK2 and TAp73 interaction, confirmed by co-immunoprecipitation, was attenuated by CK2 inhibitor, or a T27A point-mutation of this predicted CK2 threonine phospho-acceptor site of TAp73. Further, T27A mutation attenuated phosphorylation, while enhancing TAp73 function in repressing CSC gene expression and SP cells. A new CK2 inhibitor, CX-4945, inhibited CSC related SP cells, clonogenic survival, and spheroid formation. Our study unveils a novel regulatory mechanism whereby aberrant CK2 signaling inhibits TAp73 to promote the expression of CSC genes and phenotype.
Virus Research, 1996
The complete nucleotide sequence of the RNA 3 of hydrangea mosaic virus (HdMV) was determined. It... more The complete nucleotide sequence of the RNA 3 of hydrangea mosaic virus (HdMV) was determined. It consists of 2268 nucleotides and contains two open reading frames (ORF). ORF 1 encodes for a putative translation product of 293 amino acids which shared 64.9% identity with the 3a protein of tobacco streak virus (TSV). ORF 2 encodes for a putative translation product of 220 amino acids which shared 54.2% identity with the coat protein of TSV. The relationship between the proteins of HdMV and the corresponding proteins of ilarviruses other than TSV was more distant. No zinc-finger-like motif was found in the coat protein of HdMV but the N-terminus of the protein was rich in basic amino acids. Both terminal, non-coding regions of HdMV RNA 3 contained repeated sequences with corresponding homologous fragments in the RNA 3 of TSV. On the basis of the similarities between HdMV and TSV that we detected, we propose that HdMV be included in subgroup 1 of the genus Ilarvirus together with TSV.
Nature Biotechnology, 2010
Protein-Based Aqueous−Multiphasic Systems
Langmuir, 2010
This paper reports the formation of aqueous-multiphasic systems (AMPS) exclusively made using ela... more This paper reports the formation of aqueous-multiphasic systems (AMPS) exclusively made using elastin-like polypeptides (ELP) which have the ability to undergo reversible inverse phase transitions. Manipulating variables such as the salt concentration and the molecular weight and the composition of ELPs (using different amino acid sequences or by fusing the ELP with different functional proteins) permits modulation of the temperature at which phase transition takes place, the number of phases that are formed, and the composition of the multiple aqueous phases. Using these variables, isotropic hybrid colloids with tunable functionality (in this case, fluorescent intensity) and anisotropic colloids with variable morphologies could be generated. While formation of AMPS and anisotropic colloids has been reported in the literature using synthetic polymers, to our knowledge this is the first report of generating such systems using proteins.
Self-Cleavable Stimulus Responsive Tags for Protein Purification without Chromatography
Journal of the American Chemical Society, 2005
A simple method to purify recombinant proteins is described by fusing a target protein with an in... more A simple method to purify recombinant proteins is described by fusing a target protein with an intein and an elastin-like polypeptide that only requires NaCl, dithiothreitol, and a syringe filter to isolate the target protein from Escherichia coli lysate. This tripartite fusion system enables rapid isolation of the target protein without the need for affinity chromatography for purification or proteases for cleavage of the target protein from the fusion. The elastin-like polypeptide tag imparts reversible phase transition behavior to the tripartite fusion so that the fusion protein can be selectively aggregated in cell lysate by the addition of NaCl. The aggregates are isolated by microfiltration and resolubilized by reversal of the phase transition in low ionic strength buffer. After resolubilizing the fusion protein, the intein is activated to cleave the target protein from the elastin-intein tag, and the target protein is then isolated from the elastin-intein fusion by an additional phase transition cycle.
In vivo and in vitro application of elastin-like polypeptides
Elastin-like polypeptides (ELP) are artificially designed protein biopolymers that can be produce... more Elastin-like polypeptides (ELP) are artificially designed protein biopolymers that can be produced by living organisms. These proteins have the unique ability to undergo reversible inverse phase transition, in response to changes in temperature and/or addition of chaotropic salts. Below the transition temperature (Tt), ELP is soluble in water. Increasing the temperature above Tt, ELP coacervates into an aqeous ELP-rich phase. In this thesis, this unique feature of ELP was used in for recombinant protein purification and for the formation of aqueous multiple-phase systems. ^ For protein purification, ELP was fused with an intein and a model protein (thioredoxin), to demonstrate a simple and inexpensive approach for recombinant protein purification. The ELP tags replace the chromatographic media and the intein replaces the use of the protease in conventional methods. Using ELP tags was found to be consistent with large-scale recombinant protein production/purification by purifying an ...
The complete nucleotide sequence of RNA 3 of citrus leaf rugose and citrus variegation ilarviruses
Journal of General Virology, 1995
The complete nucleotide sequence of the RNA 3 of lilac ring mottle ilarvirus
Journal of General Virology, 1995
In vivo formation of protein based aqueous microcompartments
Journal of the American Chemical Society, 2009
In this paper, we report the formation of protein based liquid droplets resulting in the formatio... more In this paper, we report the formation of protein based liquid droplets resulting in the formation of in vivo microcompartments in E. coli or tobacco cells. These microcompartments were generated by expressing elastin-like polypeptides (ELP), which have the ability to undergo a reversible phase transition, resulting in the formation of an aqueous two-phase system (ATPS) in the cytoplasm of the cell. We prove that these microcompartments are liquid by expressing a fusion protein consisting of ELP and GFP and by performing fluorescence recovery after photobleaching (FRAP) experiments at different stages of cell cultivation. In the initial phases of cell growth, the fusion protein concentration is low and is not sufficient to drive the formation of a second aqueous phase. As the intracellular fusion protein concentration increases with longer cultivation time, droplets start forming, and as protein expression continues, the droplets coalesce at the poles of the E. coli cells. FRAP expe...
Highly efficient synthesis of ordered mesoporous silica materials with controllable microporosity using surfactant mixtures as templates
Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2006
Ordered mesoporous silica materials (SBA-family) with 2-D hexagonal (p6mm) and 3-D cubic (Im3m) s... more Ordered mesoporous silica materials (SBA-family) with 2-D hexagonal (p6mm) and 3-D cubic (Im3m) symmetry have been efficiently synthesized by using the mixture of triblock copolymer surfactant and sodium dodecylsulfonate as co-templates at relatively low ...
Chinese Science Bulletin, 2011
Uropathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary trac... more Uropathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary tract infection (UTI). Several UPEC virulence factors have been identified, but more are yet to be found. We previously identified a novel 789-bp-long DNA fragment (named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique. In the present study, we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity. Through bioinformatic analysis, we predicted that this gene is a 1311-bp open reading frame (ORF), which we designated ORF R049 (GenBank accession No.: EF488001). We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography. Using mouse immunosera generated by the purified protein, we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting. To test the potential of this protein as a vaccine candidate, we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract. The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group. However, the degree of renal pathological damage was not significantly improved in the immunized mice. Our study has identified a novel gene of UPEC which can generate protective immunity against UTI. This novel gene provides a promising new vaccine candidate. uropathogenic Escherichia coli, genome walking, cloning and expression, immunoprotection
Purification of an elastin-like fusion protein by microfiltration
Biotechnology and Bioengineering, 2006
This article describes a simple and potentially scalable microfiltration method for purification ... more This article describes a simple and potentially scalable microfiltration method for purification of recombinant proteins. This method is based on the fact that when an elastin-like polypeptide (ELP) is fused to a target protein, the inverse phase transition behavior of the ELP tag is imparted to the fusion protein. Triggering the phase transition of a solution of the ELP fusion protein by an increase in temperature, or isothermally by an increase in salt concentration, results in the formation of micron-sized aggregates of the ELP fusion protein. In this article, it is shown that these aggregates are efficiently retained by a microfiltration membrane, while contaminating E. coli proteins passed through the membrane upon washing. Upon reversing the phase transition by flow of Milli-Q water, soluble, pure, and functionally active protein is eluted from the membrane. Proof-of principle of this approach was demonstrated by purifying a fusion of thioredoxin with ELP (Trx-ELP) with greater than 95% recovery of protein and with greater than 95% purity (as estimated from SDS-PAGE gels). The simplicity of this method is demonstrated for laboratory scale purification by purifying Trx-ELP from cell lysate using a syringe and a disposable microfiltration cartridge. The potential scalability of this purification as an automated, continuous industrial-scale process is also demonstrated using a continuous stirred cell equipped with a microfiltration membrane.
Rapid construction and characterization of synthetic antibody libraries without DNA amplification
Biotechnology and Bioengineering, 2010
We report on a simple method to rapidly generate very large libraries of genes encoding mutant pr... more We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.
Biomacromolecules, 2006
In this paper, we demonstrate proof-of-principle for a method that allows selective recovery of m... more In this paper, we demonstrate proof-of-principle for a method that allows selective recovery of molecules present at very low concentrations in complex mixtures. The method makes use of an elastin-like polypeptide (ELP) as a coaggregant for the capture of an ELP tagged recombinant protein present at concentrations as low as 10 pM, with a recovery higher than 90%. This coaggregation process was found to be independent of the concentration, at least up to 10 pM concentration of the ELP tagged protein. The coaggregation process is highly specific as was demonstrated by spiking crude cell lysate with the ELP tagged recombinant protein to a final concentration of 1 nM and recovering more than 80% of it to a high level of purity. The method should be particularly useful for high-throughput proteomic studies, where small amounts of poorly expressed proteins could be recovered for analysis by mass spectrometry. In a more general context, the concept presented in this paper provides a method that is highly efficient, specific, and fully reversible, which should render it useful in areas other than recombinant protein purification.
Archives of Virology, 2003
ACS Applied Materials & Interfaces, 2014
Morphological evolution associated with silicidation of Co thin films deposited on (100) and (1 1... more Morphological evolution associated with silicidation of Co thin films deposited on (100) and (1 1 1) Si substrates has been followed using tra~lsmission electron microscopy with in situ thermal annealing from ambient temperature up to 850°C. Noticeable structural changes dssociated with the formation of CoSi, occur at temperatures as low as 400°C and the reaction is essentially complete at about 500°C. Prolonged heating above 500°C leads to CoSi, grain growth and coalescence and, finally, to pinholes formatio11. Silicidation of Co films on (100) and (1 11) Si substrates follo~vs the same pattern. The morphology of films annealed in situ is similar to those annealed ex situ except that the SilCoSi2 interface appears to be much rougher. This behavior is associated with the specific geometry of cross-sectional TEM specimens, where surface diffusion dominates bulk diffusion. Very thin Co films, which have less contribution from surface diffusion than thicker films, are ideal for studying dynamic phenomena at CoISi reactive interfaces.
XPS and AES investigation of nanometer composite coatings of NiPZnX on steel surface (ZnX = ZnSnO3, Zn3(PO4)2, ZnSiO3)
Applied Surface Science, 2001
In this paper, the physical chemistry nature of the NiPZnX coatings have been studied by weighi... more In this paper, the physical chemistry nature of the NiPZnX coatings have been studied by weighing method, accelerated corrosion tests, tarnish test, high-temperature oxidation tests, Auger electron spectroscopy (AES) and X-ray photoelectron spectroscopy (XPS). The result shows that the surfaces of the coatings are homogeneous, polished, solid and with strong corrosion resistance.