A New Versatile Immobilization Tag Based on the Ultra High Affinity and Reversibility of the Calmodulin-Calmodulin Binding Peptide Interaction.

Reversible, high-affinity immobilization tags are critical tools for myriad biological applications. However, inherent issues are associated with a number of the current methods of immobilization. Particularly, a critical element in phage display sorting is functional immobilization of target proteins. To circumvent these problems, we have used a mutant (N5A) of calmodulin binding peptide (CBP) as an immobilization tag in phage display sorting. The immobilization relies on the ultra high affinity of calmodulin to N5A mutant CBP (RWKKNFIAVSAANRFKKIS) in presence of calcium (KD~2 pM), which can be reversed by EDTA allowing controlled "capture and release" of the specific binders. To evaluate the capabilities of this system, we chose eight targets, some of which were difficult to overexpress and purify with other tags and some had failed in sorting experiments. In all cases, specific binders were generated using a Fab phage display library with CBP-fused constructs. KD values of the Fabs were in subnanomolar to low nanomolar (nM) ranges and were successfully used to selectively recognize antigens in cell-based experiments. Some of these targets were problematic even without any tag; thus, the fact that all led to successful selection endpoints means that borderline cases can be worked on with a high probability of a positive outcome. Taken together with examples of successful case specific, high-level applications like generation of conformation-, epitope- and domain-specific Fabs, we feel that the CBP tag embodies all the attributes of covalent immobilization tags but does not suffer from some of their well-documented drawbacks.

[1]  L. O. Penalva,et al.  Biotinylated tags for recovery and characterization of ribonucleoprotein complexes. , 2004, BioTechniques.

[2]  T. Lybrand,et al.  Streptavidin-biotin binding energetics. , 1999, Biomolecular engineering.

[3]  R. Ward,et al.  Retrieval of human antibodies from phage-display libraries using enzymatic cleavage. , 1996, Journal of immunological methods.

[4]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[5]  Marjeta Urh,et al.  HaloTag: a novel protein labeling technology for cell imaging and protein analysis. , 2008, ACS chemical biology.

[6]  J. Butler,et al.  The physical and functional behavior of capture antibodies adsorbed on polystyrene. , 1992, Journal of immunological methods.

[7]  Zhifeng Shao,et al.  Fc-fusion proteins: new developments and future perspectives , 2012, EMBO molecular medicine.

[8]  Kathryn S. Prickett,et al.  A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification , 1988, Bio/Technology.

[9]  G. Winter,et al.  Phage antibodies: filamentous phage displaying antibody variable domains , 1990, Nature.

[10]  S. Sidhu,et al.  Phage display for selection of novel binding peptides. , 2000, Methods in enzymology.

[11]  F. Niesen,et al.  The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability , 2007, Nature Protocols.

[12]  K. Terpe.  Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems , 2002, Applied Microbiology and Biotechnology.

[13]  Shohei Koide,et al.  High-affinity single-domain binding proteins with a binary-code interface , 2007, Proceedings of the National Academy of Sciences.

[14]  S. Mukherjee,et al.  Expression, purification, crystallization and preliminary X-ray diffraction studies of phosphoglycerate kinase from methicillin-resistant Staphylococcus aureus MRSA252. , 2011, Acta crystallographica. Section F, Structural biology and crystallization communications.

[15]  Volker A Erdmann,et al.  The Nano-tag, a streptavidin-binding peptide for the purification and detection of recombinant proteins. , 2004, Protein expression and purification.

[16]  F. Quiocho,et al.  Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis. , 1992, Biochemistry.

[17]  Anthony D. Keefe,et al.  One-step purification of recombinant proteins using a nanomolar-affinity streptavidin-binding peptide, the SBP-Tag. , 2001, Protein expression and purification.

[18]  B. Kay,et al.  High-throughput biotinylation of proteins. , 2009, Methods in molecular biology.

[19]  A. Kossiakoff,et al.  Allosteric Control of Ligand Binding Affinity Using Engineered Conformation-Specific Effector Proteins , 2010, Nature Structural &Molecular Biology.

[20]  M. Wilchek,et al.  [14] Protein biotinylation , 1990 .

[21]  D. Beckett,et al.  A minimal peptide substrate in biotin holoenzyme synthetase‐catalyzed biotinylation , 2008, Protein science : a publication of the Protein Society.

[22]  A. Koide,et al.  Generating conformation-specific synthetic antibodies to trap proteins in selected functional states. , 2013, Methods.

[23]  Song Tan,et al.  Comparison of affinity tags for protein purification. , 2005, Protein expression and purification.

[24]  C. Tomomori-Sato,et al.  Immunoaffinity purification of protein complexes from Mammalian cells. , 2013, Methods in molecular biology.

[25]  T. Craig,et al.  Calmodulin and calmodulin-regulated protein kinases as transducers of intracellular calcium signals. , 1988, Cold Spring Harbor symposia on quantitative biology.

[26]  O. Hantschel,et al.  Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains , 2013, Proceedings of the National Academy of Sciences.

[27]  S. Sidhu,et al.  Synthetic antibodies: concepts, potential and practical considerations. , 2012, Methods.

[28]  O. Laitinen,et al.  Brave new (strept)avidins in biotechnology. , 2007, Trends in biotechnology.

[29]  A. Koide,et al.  Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor. , 2006, Protein expression and purification.

[30]  Mati Cohen,et al.  Noncovalent, site-specific biotinylation of histidine-tagged proteins. , 2007, Analytical chemistry.

[31]  Somnath Mukherjee,et al.  Crystal structure of glyceraldehyde-3-phosphate dehydrogenase 1 from methicillin-resistant Staphylococcus aureus MRSA252 provides novel insights into substrate binding and catalytic mechanism. , 2010, Journal of molecular biology.

[32]  Michael J Taussig,et al.  Double-hexahistidine tag with high-affinity binding for protein immobilization, purification, and detection on ni-nitrilotriacetic acid surfaces. , 2006, Analytical chemistry.

[33]  D. Christ,et al.  Selection of human antibody fragments by phage display , 2007, Nature Protocols.

[34]  John B Wojcik,et al.  Accelerating phage-display library selection by reversible and site-specific biotinylation. , 2009, Protein engineering, design & selection : PEDS.

[35]  D. Ricklin,et al.  Oligohis‐tags: mechanisms of binding to Ni2+‐NTA surfaces , 2009, Journal of molecular recognition : JMR.

[36]  T. Kunkel.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[37]  A. Chapman-Smith,et al.  Molecular biology of biotin attachment to proteins. , 1999, The Journal of nutrition.

[38]  A. Koide,et al.  T Cell Receptor-Like Recognition of Tumor In Vivo by Synthetic Antibody Fragment , 2012, PloS one.

[39]  G. Winter,et al.  Calmodulin as a Versatile Tag for Antibody Fragments , 1995, Bio/Technology.

[40]  Frederic A. Fellouse,et al.  High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. , 2007, Journal of molecular biology.

[41]  D. Neri,et al.  Alanine substitutions in calmodulin-binding peptides result in unexpected affinity enhancement. , 1996, Journal of molecular biology.

[42]  J. Mccoy,et al.  A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli. , 1998, Nucleic acids research.