TY - JOUR
T1 - Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand
AU - Wllberg, Helena
AU - Löfdahl, Per Ke
AU - Tschapalda, Kirsten
AU - Uhlén, Mathias
AU - Tolmachev, Vladimir
AU - Nygren, Per Ke
AU - Sthl, Stefan
N1 - Funding Information:
This research was supported by grants from VINNOVA ( P35722-1 ) and the Swedish Foundation for Strategic Research ( RBa08-0067 ) (Stiftelsen för Strategisk Forskning). The authors wish to express their gratitude to Prof. Sophia Hober for productive discussions and to Dr. Tove Alm , Johanna Steen , and Joel Lindgren, KTH , for technical assistance.
Copyright:
Copyright 2011 Elsevier B.V., All rights reserved.
PY - 2011/3
Y1 - 2011/3
N2 - Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these ZHER2 affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ß-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a ZHER2 affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of ZHER2. This novel anti-idiotypic affibody molecule ZE01 was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of ZHER2 affibody molecules, having a common binding surface for HER2. Eight such ZHER2 affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (99mTc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.
AB - Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these ZHER2 affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ß-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a ZHER2 affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of ZHER2. This novel anti-idiotypic affibody molecule ZE01 was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of ZHER2 affibody molecules, having a common binding surface for HER2. Eight such ZHER2 affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (99mTc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.
KW - Affibody molecule
KW - Affinity column
KW - Anti-idiotypic affibody ligand
KW - Molecular imaging
KW - Protein engineering
KW - Protein fragment complementation assay
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U2 - 10.1016/j.pep.2010.10.008
DO - 10.1016/j.pep.2010.10.008
M3 - Article
C2 - 21029777
AN - SCOPUS:78650180317
VL - 76
SP - 127
EP - 135
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
IS - 1
ER -