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Howard A Chase - One of the best experts on this subject based on the ideXlab platform.

  • intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding
    Biotechnology Progress, 2006
    Co-Authors: Matthew H Hutchinson, Howard A Chase
    Abstract:

    : This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the Polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other Polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.

  • removal of poly histidine fusion tags from recombinant proteins purified by expanded bed adsorption
    Biotechnology and Bioengineering, 2005
    Co-Authors: Norhafizah Abdullah, Howard A Chase
    Abstract:

    Enzymatic methods have been used to cleave the C- or N-terminus Polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus Polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal Polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His6 by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His6 to MBP was 16 h, as judged by SDS–PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using “on-column” tag digestion. On-column tag digestion involves cleavage of histidine tag from Polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal Polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of Polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions. © 2005 Wiley Periodicals, Inc.

  • removal of poly histidine fusion tags from recombinant proteins purified by expanded bed adsorption
    Biotechnology and Bioengineering, 2005
    Co-Authors: Norhafizah Abdullah, Howard A Chase
    Abstract:

    Enzymatic methods have been used to cleave the C- or N-terminus Polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus Polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal Polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from Polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal Polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of Polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.

Hedi Mattoussi - One of the best experts on this subject based on the ideXlab platform.

  • kinetics of metal affinity driven self assembly between proteins or peptides and cdse zns quantum dots
    Journal of Physical Chemistry C, 2007
    Co-Authors: Kim E Sapsford, Igor L Medintz, Thomas Pons, Seiichiro Higashiya, Florence M Brunel, And Philip E Dawson, Hedi Mattoussi
    Abstract:

    We present a molecular characterization of metal-affinity driven self-assembly between CdSe−ZnS core−shell quantum dots (QDs) and a series of proteins and peptides appended with various length Polyhistidine tags. In particular, we investigated the kinetics of self-assembly between surface-immobilized QDs and proteins/peptides under flow conditions, as well as between freely diffusing QDs and proteins/peptides (solution phase). In the first configuration, QDs were immobilized onto functionalized substrates and then exposed to dye-labeled peptides/proteins. Using evanescent wave excitation, we assessed self-assembly by monitoring the time-dependent changes in the dye fluorescence. In solution, the kinetics of self-assembly was monitored via energy transfer between QDs and dye-labeled proteins/peptides. These measurements allowed determination of the kinetic parameters, including the association and dissociation rates (kon and koff) and the apparent binding constant (Kd). We find that self-assembly is rapid ...

  • a reactive peptidic linker for self assembling hybrid quantum dot dna bioconjugates
    Nano Letters, 2007
    Co-Authors: Igor L Medintz, Lorenzo Berti, Thomas Pons, Amy F Grimes, Douglas S English, Andrea Alessandrini, Paolo Facci, Hedi Mattoussi
    Abstract:

    Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe−ZnS core−shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD−D...

Ugur Sahin - One of the best experts on this subject based on the ideXlab platform.

  • Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide
    Journal of nanobiotechnology, 2018
    Co-Authors: Jens Schumacher, Tijana Bacic, René Staritzbichler, Matin Daneschdar, Thorsten Klamp, Philipp Arnold, Sabrina Jägle, Özlem Türeci, Jürgen Markl, Ugur Sahin
    Abstract:

    Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. The effects of a carboxy-terminal Polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. These findings establish the genetically simply transferable C-terminal Polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.

  • Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide
    BMC, 2018
    Co-Authors: Jens Schumacher, Tijana Bacic, René Staritzbichler, Matin Daneschdar, Thorsten Klamp, Philipp Arnold, Sabrina Jägle, Özlem Türeci, Jürgen Markl, Ugur Sahin
    Abstract:

    Abstract Background Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. Results The effects of a carboxy-terminal Polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. Conclusions These findings establish the genetically simply transferable C-terminal Polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability

Norhafizah Abdullah - One of the best experts on this subject based on the ideXlab platform.

  • removal of poly histidine fusion tags from recombinant proteins purified by expanded bed adsorption
    Biotechnology and Bioengineering, 2005
    Co-Authors: Norhafizah Abdullah, Howard A Chase
    Abstract:

    Enzymatic methods have been used to cleave the C- or N-terminus Polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus Polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal Polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His6 by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His6 to MBP was 16 h, as judged by SDS–PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using “on-column” tag digestion. On-column tag digestion involves cleavage of histidine tag from Polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal Polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of Polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions. © 2005 Wiley Periodicals, Inc.

  • removal of poly histidine fusion tags from recombinant proteins purified by expanded bed adsorption
    Biotechnology and Bioengineering, 2005
    Co-Authors: Norhafizah Abdullah, Howard A Chase
    Abstract:

    Enzymatic methods have been used to cleave the C- or N-terminus Polyhistidine tags from histidine tagged proteins following expanded bed purification using immobilized metal affinity chromatography (IMAC). This study assesses the use of Factor Xa and a genetically engineered exopeptidase dipeptidyl aminopeptidase-1 (DAPase-1) for the removal of C-terminus and N-terminus Polyhistidine tags, respectively. Model proteins consisting of maltose binding protein (MBP) having a C- or N-terminal Polyhistidine tag were used. Digestion of the hexahistidine tag of MBP-His(6) by Factor Xa and HT15-MBP by DAPase-1 was successful. The time taken to complete the conversion of MBP-His(6) to MBP was 16 h, as judged by SDS-PAGE and Western blots against anti-His antibody. When the detagged protein was purified using subtractive IMAC, the yield was moderate at 71% although the overall recovery was high at 95%. Likewise, a yield of 79% and a recovery of 97% was obtained when digestion was performed with using "on-column" tag digestion. On-column tag digestion involves cleavage of histidine tag from Polyhistidine tagged proteins that are still bound to the IMAC column. Digestion of an N-terminal Polyhistidine tag from HT15-MBP (1 mg/mL) by the DAPase-I system was superior to the results obtained with Factor Xa with a higher yield and recovery of 99% and 95%, respectively. The digestion by DAPase-I system was faster and was complete at 5 h as opposed to 16 h for Factor Xa. The detagged MBP proteins were isolated from the digestion mixtures using a simple subtractive IMAC column procedure with the detagged protein appearing in the flowthrough and washing fractions while residual dipeptides and DAPase-I (which was engineered to exhibit a poly-His tail) were adsorbed to the column. FPLC analysis using a MonoS cation exchanger was performed to understand and monitor the progress and time course of DAPase-I digestion of HT15-MBP to MBP. Optimization of process variables such as temperature, protein concentration, and enzyme activity was developed for the DAPase-I digesting system on HT15-MBP to MBP. In short, this study proved that the use of either Factor Xa or DAPase-I for the digestion of Polyhistidine tags is simple and efficient and can be carried out under mild reaction conditions.

Jens Schumacher - One of the best experts on this subject based on the ideXlab platform.

  • Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide
    Journal of nanobiotechnology, 2018
    Co-Authors: Jens Schumacher, Tijana Bacic, René Staritzbichler, Matin Daneschdar, Thorsten Klamp, Philipp Arnold, Sabrina Jägle, Özlem Türeci, Jürgen Markl, Ugur Sahin
    Abstract:

    Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. The effects of a carboxy-terminal Polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. These findings establish the genetically simply transferable C-terminal Polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability.

  • Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide
    BMC, 2018
    Co-Authors: Jens Schumacher, Tijana Bacic, René Staritzbichler, Matin Daneschdar, Thorsten Klamp, Philipp Arnold, Sabrina Jägle, Özlem Türeci, Jürgen Markl, Ugur Sahin
    Abstract:

    Abstract Background Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. Results The effects of a carboxy-terminal Polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. Conclusions These findings establish the genetically simply transferable C-terminal Polyhistidine-peptide as a general stabilizing element for chimeric HBcAg-VLPs to increase their suitability