Engineering knottins as novel binding agents.
Cystine-knot miniproteins, also known as knottins, contain a conserved core of three tightly woven disulfide bonds which impart extraordinary thermal and proteolytic stability. Interspersed between their conserved cysteine residues are constrained loops that possess high levels of sequence diversity among knottin family members. Together these attributes make knottins promising molecular scaffolds for protein engineering and translational applications. While naturally occurring knottins have shown potential as both diagnostic agents and therapeutics, protein engineering is playing an important and increasing role in creating designer molecules that bind to a myriad of biomedical targets. Toward this goal, rational and combinatorial approaches have been used to engineer knottins with novel molecular recognition properties. Here, methods are described for creating and screening knottin libraries using yeast surface display and fluorescence-activated cell sorting. Protocols are also provided for producing knottins by synthetic and recombinant methods, and for measuring the binding affinity of knottins to target proteins expressed on the cell surface.
Department of Bioengineering, Cancer Institute, and Bio-X Program, Stanford University, Stanford, California, USA.
Sarah J
SJ
Moore
Jennifer R
JR
Cochran
D000345:Affinity Labels / Q000737:chemistry; D000818:Animals; D002352:Carrier Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003545:Cysteine / Q000737:chemistry; D057771:Cystine-Knot Miniproteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004247:DNA / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D016366:Open Reading Frames; D019151:Peptide Library; D010843:Pichia / Q000737:chemistry; D010957:Plasmids / Q000737:chemistry; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011956:Receptors, Cell Surface / Q000737:chemistry; D011994:Recombinant Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification; D060327:Solid-Phase Synthesis Techniques; D013379:Substrate Specificity; D015003:Yeasts / Q000737:chemistry
D000345:Affinity Labels; D002352:Carrier Proteins; D057771:Cystine-Knot Miniproteins; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D019151:Peptide Library; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D004247:DNA; D003545:Cysteine