| 1. |
- Bandmann, Nina, et al.
(author)
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Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins
- 2002
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In: Journal of Biotechnology. - 0168-1656 .- 1873-4863. ; 93:1, s. 1-14
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Journal article (peer-reviewed)abstract
- Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated, Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M 13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.
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| 2. |
- Becker, Kristian, et al.
(author)
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Multipurpose peptide tags for protein isolation.
- 2008
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In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1202:1, s. 40-46
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Journal article (peer-reviewed)abstract
- A multifunctional peptide tag (HYDHYD) consisting of histidine, tyrosine and aspartate residues was fused to the N-terminal ends of green fluorescent protein (GFP), lactate dehydrogenase (LDH) and human hemoglobin (Hb), proteins which were subjected to ion-exchange chromatography (IEC), aqueous two-phase system partition, immobilized metal-ion affinity chromatography (IMAC), and hydrophobic interaction chromatography (HIC). Tagged GFP was retained significantly longer (>1 column volume) in both HIC and IEC. It exhibited 3x greater partition in favor of the hydrophobic phase in a two-phase system and 96% could be bound to an IMAC column which did not bind native GFP.
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| 3. |
- Fexby, Sara, et al.
(author)
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Novel in situ polymerized coatings for hydrophobic interaction chromatography media
- 2007
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In: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1161:1-2, s. 234-241
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Journal article (peer-reviewed)abstract
- Hydrophobic interaction chromatography (HIC) and other capture media are typically produced by grafting different ligands to base matrices at defined surface densities. This often complicates media production. An alternative approach to media involving in situ radical initiated polymerization was used to graft polymer coatings directly at Sepharose(D polymeric base matrices. This method appears suitable for producing many different chromatography media on a variety of base matrices. In the present study, it also favorably increased the solution pressure-flow properties of a Sepharose base matrix used to produce HIC media. A wide range of HIC media could be produced by simply varying the reaction ratio of butyl vinyl ether, and hydroxybutyl vinyl ether. The new HIC media was evaluated using five test proteins (bovine serum albumin, ribonuclease A, (x-chymotrypsinogen A, myoglobin and (x-lactalbumin). The media exhibited classic HIC behavior, predictably controlled hydrophobicity, plus good protein selectivity, capacity (70 mg protein/ml gel) and often total protein recovery. By modifying the degree of matrix hydrophobicity, we could also reduce effects of protein denaturation often seen with conventional HIC and observed as multiple peaks in the chromatograms. Separation of crude protein extracts from Eschericha coli, expressing a green fluorescent protein (GFPuv) and, a more hydrophobic, recombinantly-modified, tyrosine-tagged green fluorescent protein (Y-PYPY-GFPuv), was also performed. These proteins were very stable, exhibited significantly different retention times, and could be used to study the ability of the media to work with complex protein mixtures. Such GFP mutants appear ideal for characterizing the performance of chromatographic media. (c) 2007 Published by Elsevier B.V.
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| 4. |
- Johansson, Hans-Olof, et al.
(author)
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Modeling of protein interactions with surface-grafted charged polymers. Correlations between statistical molecular modeling and a mean field approach
- 2006
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In: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 22:21, s. 8920-8930
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Journal article (peer-reviewed)abstract
- Ion exchange media involving charge groups attached to flexible polymers are widely used for protein purification. Such media often provide enhanced target protein purity and yield. Yet, little is understood about protein interaction with such media at the molecular level, or how different media architectures might affect separation performance. To gain a better understanding of such adsorptive systems, statistical mechanical perturbation calculations, utilizing a Debye-Huckel potential, were performed on surface-grafted charged polymers and their interaction with model proteins. The studied systems were weakly charged, and the polymers were linear and relatively short (degree of polymerization is 30). Segment distributions from the surface were also determined. The interaction of spherical model protein particles of 12-30 angstrom radius were investigated with respect to polymer grafting density, distance from matrix surface, protein charge, and ionic strength. The partitioning coefficient of the model proteins was determined for different distances from the surface. An empirical mean field theory that scales the entropy of the protein with the square of the protein radius correlates well to Monte Carlo statistical modeling results. Upon adsorption to the polymer layers, the model proteins exhibit a critical surface charge density that is proportional to the ionic strength, independent of the grafting density, and appears to be a fundamental determinant of protein adsorption. Partitioning of protein-like nanoparticles to the charged polymer surface is only favored above the particle critical charge density.
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| 5. |
- McCann, K. B., et al.
(author)
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Polyacrylic acid based plasma fractionation for the production of albumin and IgG : Compatibility with existing commercial downstream processes
- 2020
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In: Biotechnology and Bioengineering. - : Wiley. - 0006-3592 .- 1097-0290. ; 117:4, s. 1072-1081
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Journal article (peer-reviewed)abstract
- Commercial fractionation of human plasma into immunoglobulin- and albumin-rich fractions is often initiated with sequential cold ethanol-based precipitation methods, which have changed little over the past 70 years. The required low temperature (−4 to −8°C) and high concentrations of ethanol 8–40%) necessitate large-scale fixed processing lines, and major capital investment and operating costs. The resulting fractions are then further purified by ethanol based precipitation or chromatographic procedures to obtain the purified final product. Aqueous polyacrylic acid (PAA) based precipitation, which readily interfaces with existing downstream processing, could offer advantages with respect to cost, safety, environmental impact, and flexibility. Sequential precipitation with 7%, 12%, and 20% (w/v) solutions of PAA 8000 in the presence of a kosmotropic salt (sodium citrate) gave fibrinogen-, immunoglobulin-, and albumin-rich fractions with 80–90% yield and 64%, 55%, and 82% purity, respectively. Further purification of the IgG-rich precipitate by caprylic acid precipitation and anion exchange chromatography, achieved a target purity of >99%. This was also achieved for the downstream processing of the albumin-rich precipitate using a two-step ion exchange chromatographic procedure. This work shows that PAA precipitation can be used in place of cold ethanol precipitation to generate crude IgG and albumin fractions which can be purified to final products of acceptable purity.
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| 6. |
- Rodrigo, Gustav, et al.
(author)
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Antibody Fragments and Their Purification by Protein L Affinity Chromatography
- 2015
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In: ANTIBODIES. - : MDPI AG. - 2073-4468. ; 4:3, s. 259-277
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Research review (peer-reviewed)abstract
- Antibodies and related proteins comprise one of the largest and fastest-growing classes of protein pharmaceuticals. A majority of such molecules are monoclonal antibodies; however, many new entities are antibody fragments. Due to their structural, physiological, and pharmacological properties, antibody fragments offer new biopharmaceutical opportunities. In the case of recombinant full-length antibodies with suitable Fc regions, two or three column purification processes centered around Protein A affinity chromatography have proven to be fast, efficient, robust, cost-effective, and scalable. Most antibody fragments lack Fc and suitable affinity for Protein A. Adapting proven antibody purification processes to antibody fragments demands different affinity chromatography. Such technology must offer the unit operation advantages noted above, and be suitable for most of the many different types of antibody fragments. Protein L affinity chromatography appears to fulfill these criteriasuggesting its consideration as a key unit operation in antibody fragment processing.
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| 7. |
- Soares, R.R.G., et al.
(author)
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Partitioning in aqueous two-phase systems : Analysis of strengths, weaknesses, opportunities and threats
- 2015
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In: Biotechnology Journal. - : Wiley. - 1860-6768 .- 1860-7314. ; 10:8, s. 1158-1169
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Journal article (peer-reviewed)abstract
- For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.
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| 8. |
- Torto, N., et al.
(author)
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In situ poly(ethylene imine) coating of hollow fiber membranes used for microdialysis sampling
- 2004
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In: Pure and Applied Chemistry. - : Walter de Gruyter GmbH. - 0033-4545 .- 1365-3075. ; 76:4, s. 879-888
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Journal article (peer-reviewed)abstract
- A method for the in situ modification of hollow fiber membranes used as sampling units for microdialysis probes is presented. The method consists of adsorption-coating, high-molecular-weight poly(ethylene imine), PEI, onto membranes, already fitted on microdialysis probes. Modification of membranes was designed to specifically explore the so-called Andrade effects and thus enhance the interaction of membranes with enzyme. The performances of polysulfone, polyethersulfone, and polyamide membranes modified with PEI-enzyrne complex were evaluated based on the membrane extraction fraction for maltose and maltotriose and membrane morphology as examined by scanning electron microscopy. Of the membranes tested, the PEI-enzyme complex least affected the performance of the polysulfone membranes. Conversion of maltoheptaose to maltotriose and maltose was increased reproducibly (within a 5 % relative standard deviation) by 50 % for modified membranes compared to the native hollow fiber membranes. The results demonstrate the potential to effectively modify membranes already fitted on a microdialysis probe. Such a procedure can be modified and employed to either promote or reduce membrane-protein interaction for hollow fibers used as microdialysis sampling units or other similar membrane applications.
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