The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment.
The facile detection and purification of a recombinant protein without detailed knowledge about its individual biochemical properties constitutes a problem of general interest in protein engineering. The use of a novel kind of random peptide library for the stepwise engineering of a C-terminal fusion peptide which confers binding activity towards streptavidin is described in this study. Because of its widespread use as part of a variety of conjugates and other affinity reagents, streptavidin constitutes the binding partner of choice both for detection and purification purposes. The streptavidin-affinity tag was engineered at the C-terminus of the VH domain as part of the D1.3 Fv fragment which was functionally expressed in Escherichia coli. Irrespective of whether it was displayed by the VH or the VL domain, the optimized version of the affinity peptide termed 'Strep-tag' allowed the detection of the Fv fragment both on Western blots and in ELISAs by a streptavidin-alkaline phosphatase conjugate. In addition, the one-step purification of the intact Fv fragment carrying a single Strep-tag at the C-terminus of only one of its domains was achieved by affinity chromatography with streptavidin-agarose using very mild elution conditions.
Schmidt
T G
TG
Max-Planck-Institut für Biophysik, Frankfurt/Main, Germany.
Skerra
A
A
D000345:Affinity Labels / Q000378:metabolism; D000469:Alkaline Phosphatase; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D016384:Consensus Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D016259:Genes, myc; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D019809:Streptavidin
D000345:Affinity Labels; D001426:Bacterial Proteins; D007128:Immunoglobulin Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; D019809:Streptavidin; D000469:Alkaline Phosphatase; D009113:Muramidase