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1
Content available remote One-step purification of vitronectin from human plasma by affinity chromatography on phage-displayed peptides
100%
Skurzynski S.
Acta Biochimica Polonica
|
2010
|
tom 57
|
nr 1
89-93
EN
A novel affinity purification method for rapid isolation of vitronectin (VN) from human plasma is described. Recently we have used phage display technology to obtain clones expressing peptides with high binding activity for VN. The isolated "strong VN binders" were covalently coupled to CNBr-activated Sepharose. Human plasma was applied to the column and bound VN was eluted using 0.5 M acetic acid, giving purity exceeding 90%. The developed method is a convenient alternative to conventional antibody-antigen affinity chromatography techniques for purification of VN, as it offers low ligand cost, is rapid and ensures good protein recovery from human plasma.
2
Content available remote A novel, stable, helical scaffold as an alternative binder - construction of phage display libraries
100%
Cyranka-Czaja A. , Otlewski J.
Acta Biochimica Polonica
|
2012
|
tom 59
|
nr 3
383-390
EN
Specific, high affinity binding macromolecules are of great importance for biomedical and biotechnological applications. The most popular classical antibody-based molecules have recently been challenged by alternative scaffolds with desirable biophysical properties. Phage display technology applied to such scaffolds allows generation of potent affinity reagents by in vitro selection. Here, we report identification and characterization of a novel helical polypeptide with advantageous biophysical properties as a template for construction of phage display libraries. A three-helix bundle structure, based on Measles virus phosphoprotein P shows a very favourable stability and solubility profile. We designed, constructed and characterized six different types of phage display libraries based on the proposed template. Their functional size of over 109 independent clones, balanced codon bias and decent display level are key parameters attesting to the quality and utility of the libraries. The new libraries are a promising tool for isolation of high affinity binders based on a small helical scaffold which could become a convenient alternative to antibodies.
3
Content available remote On the computational complexity of designing DNA randomizations in a combinatorial protein experiment
88%
Błażewicz J. , Dziurdza B. , Markiewicz W. T. , Oguz C.
Foundations of Computing and Decision Sciences
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2007
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tom Vol. 32, No. 3
185-197
EN
In combinatorial protein experiments based on phage display and similar methods, protein libraries are constructed by expressing a partially randomized DNA (gene) libraries. Since the distribution of proteins in the output library depends on nucleotides frequencies in DNA library one has to adjust them carefully taking into account diversity-completeness trade-off and results from possible previous cycles of experiments (i.e. knowledge about sequences that have been already obtained and tested). The approach considered in this paper allows to maximize the number of new amino acid sequences physically generated in each cycle of the experiment. The mathematical model of the described approach is presented and its computational complexity is analyzed.
4
Content available remote Degenerate specificity of PDZ domains from RhoA-specific nucleotide exchange factors PDZRhoGEF and LARG
88%
Smietana K. , Kasztura M. , Paduch M. , Derewenda U. , Derewenda Z. , Otlewski J.
Acta Biochimica Polonica
|
2008
|
tom 55
|
nr 2
269-280
EN
PDZ domains are ubiquitous protein-protein interaction modules which bind short, usually carboxyterminal fragments of receptors, other integral or membrane-associated proteins, and occasionally cytosolic proteins. Their role in organizing multiprotein complexes at the cellular membrane is crucial for many signaling pathways, but the rules defining their binding specificity are still poorly understood and do not readily explain the observed diversity of their known binding partners. Two homologous RhoA-specific, multidomain nucleotide exchange factors PDZRhoGEF and LARG contain PDZ domains which show a particularly broad recognition profile, as suggested by the identification of five diverse biological targets. To investigate the molecular roots of this phenomenon, we constructed a phage display library of random carboxyterminal hexapeptides. Peptide variants corresponding to the sequences identified in library selection were synthesized and their affinities for both PDZ domains were measured and compared with those of peptides derived from sequences of natural partners. Based on the analysis of the binding sequences identified for PDZRhoGEF, we propose a sequence for an 'optimal' binding partner. Our results support the hypothesis that PDZ-peptide interactions may be best understood when one considers the sum of entropic and dynamic effects for each peptide as a whole entity, rather than preferences for specific residues at a given position.
5
Molecular and chemical engineering of bacteriophages for potential medical applications
63%
Hodyra K. , Dabrowska K.
Archivum Immunologiae et Therapiae Experimentalis
|
2015
|
tom 63
|
nr 2
6
Cloning, expression and identification by immunohistochemistry of humanized single-chain variable fragment antibody against hepatitis C virus core protein
63%
Lun Y. , Cheng J. , Zhong Y. , Yu Z. , Wang Q. , Wang F. , Feng J.
|
|
nr 1
EN
Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library containing the gene for scFv antibody against Hepatitis C virus core protein was panned with core protein immobilized on microtiter plate wells. After five rounds of panning 60 phage clones specific to core protein were obtained and one selected clone was sequenced. It was found that the specifically detected antigen consists of 774bp and is capable of encoding 257 amino acids in the patients but not in healthy persons.
7
Degenerate specificity of PDZ domains from RhoA-specific nucleotide exchange factors PDZRhoGEF and LARG
63%
Smietana K. , Kasztura M. , Paduch M. , Derewenda U. , Derewenda Z. S. , Otlewski J.
Acta Biochimica Polonica
|
2008
|
tom 55
|
nr 2
269-280
EN
PDZ domains are ubiquitous protein–protein interaction modules which bind short, usually carboxyterminal fragments of receptors, other integral or membrane-associated proteins, and occasionally cytosolic proteins. Their role in organizing multiprotein complexes at the cellular membrane is crucial for many signaling pathways, but the rules defining their binding specificity are still poorly understood and do not readily explain the observed diversity of their known binding partners. Two homologous RhoA-specific, multidomain nucleotide exchange factors PDZRhoGEF and LARG contain PDZ domains which show a particularly broad recognition profile, as suggested by the identification of five diverse biological targets. To investigate the molecular roots of this phenomenon, we constructed a phage display library of random carboxyterminal hexapeptides. Peptide variants corresponding to the sequences identified in library selection were synthesized and their affinities for both PDZ domains were measured and compared with those of peptides derived from sequences of natural partners. Based on the analysis of the binding sequences identified for PDZRhoGEF, we propose a sequence for an ‘optimal’ binding partner. Our results support the hypothesis that PDZ–peptide interactions may be best understood when one considers the sum of entropic and dynamic effects for each peptide as a whole entity, rather than preferences for specific residues at a given position.
8
A novel, stable, helical scaffold as an alternative binder - construction of phage display libraries
63%
Cyranka-Czaja A. , Otlewski J.
|
|
nr 3
EN
 Specific, high affinity binding macromolecules are of great importance for biomedical and biotechnological applications. The most popular classical antibody-based molecules have recently been challenged by alternative scaffolds with desirable biophysical properties. Phage display technology applied to such scaffolds allows generation of potent affinity reagents by in vitro selection. Here, we report identification and characterization of a novel helical polypeptide with advantageous biophysical properties as a template for construction of phage display libraries. A three-helix bundle structure, based on Measles virus phosphoprotein P shows a very favourable stability and solubility profile. We designed, constructed and characterized six different types of phage display libraries based on the proposed template. Their functional size of over 109 independent clones, balanced codon bias and decent display level are key parameters attesting to the quality and utility of the libraries. The new libraries are a promising tool for isolation of high affinity binders based on a small helical scaffold which could become a convenient alternative to antibodies.
9
Molecular addresses of tumors: selection by in vivo phage display
63%
Li X. B. , Schluesener H. J. , Hu S. Q.
Archivum Immunologiae et Therapiae Experimentalis
|
2006
|
tom 54
|
nr 3
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