Engineering Streptavidin and a Streptavidin-Binding Peptide with Infinite Binding Affinity and Reversible Binding Capability: Purification of a Tagged Recombinant Protein to High Purity via Affinity-Driven Thiol Coupling

PLoS One. 2015 Sep 25;10(9):e0139137. doi: 10.1371/journal.pone.0139137. eCollection 2015.

Abstract

To extend and improve the utility of the streptavidin-binding peptide tag (SBP-tag) in applications ranging from affinity purification to the reversible immobilization of recombinant proteins, a cysteine residue was introduced to the streptavidin mutein SAVSBPM18 and the SBP-tag to generate SAVSBPM32 and SBP(A18C), respectively. This pair of derivatives is capable of forming a disulfide bond through the newly introduced cysteine residues. SAVSBPM32 binds SBP-tag and biotin with binding affinities (Kd ~ 10-8M) that are similar to SAVSBPM18. Although SBP(A18C) binds to SAVSBPM32 more weakly than SBP-tag, the binding affinity is sufficient to bring the two binding partners together efficiently before they are locked together via disulfide bond formation-a phenomenon we have named affinity-driven thiol coupling. Under the condition with SBP(A18C) tags in excess, two SBP(A18C) tags can be captured by a tetrameric SAVSBPM32. The stoichiometry of the disulfide-bonded SAVSBPM32-SBP(A18C) complex was determined using a novel two-dimensional electrophoresis method which has general applications for analyzing the composition of disulfide-bonded protein complexes. To illustrate the application of this reversible immobilization technology, optimized conditions were established to use the SAVSBPM32-affinity matrix for the purification of a SBP(A18C)-tagged reporter protein to high purity. Furthermore, we show that the SAVSBPM32-affinity matrix can also be applied to purify a biotinylated protein and a reporter protein tagged with the unmodified SBP-tag. The dual (covalent and non-covalent) binding modes possible in this system offer great flexibility to many different applications which need reversible immobilization capability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biotin / metabolism
  • Biotinylation
  • Carrier Proteins / metabolism*
  • Chromatography, Affinity / methods*
  • Cysteine / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Kinetics
  • Ligands
  • Maltose-Binding Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / metabolism
  • Protein Binding
  • Protein Engineering / methods*
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism*
  • Streptavidin / metabolism*
  • Sulfhydryl Compounds / metabolism*

Substances

  • Carrier Proteins
  • Ligands
  • Maltose-Binding Proteins
  • Peptides
  • Recombinant Fusion Proteins
  • Sulfhydryl Compounds
  • streptavidin-binding peptide
  • Biotin
  • Streptavidin
  • Cysteine

Grants and funding

This study was supported by Discovery grant (690573) from The Natural Sciences and Engineering Research Council of Canada (NSERC) to SLW, http://www.nserc-crsng.gc.ca/; Discovery grant (262089) from The Natural Sciences and Engineering Research Council of Canada (NSERC) to KKSN, http://www.nserc-crsng.gc.ca/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.