Highly Stable Binding Proteins Derived from the Hyperthermophilic Sso7d Scaffold

Discrete and Structurally Unique Proteins (Tāpirins) Mediate Attachment of Extremely Thermophilic Caldicellulosiruptor Species to Cellulose

“…Selected representative genes from each class of cellulolytic ta pirin (i.e. Calkro_0844 and Calkro_0845) were expressed as chimeric proteins fused to the C terminus of the yeast ␣-agglutinin protein to facilitate yeast surface display (40,54). Previously, yeast surface display systems in S. cerevisiae have successfully expressed and fused cellulose-specific CBM modules (41) and cellulases (55) from cellulolytic fungi to the yeast cell wall.…”

Inefficient Ribosomal Skipping Enables Simultaneous Secretion and Display of Proteins in Saccharomyces cerevisiae

“…The difference in signal between Sso7dstrep and Sso7dhFc is likely due to the difference in binding affinities for their respective targets. The binding affinity (K D ) of Sso7dstrep for streptavidin of 12 nM, as determined in experiments where Sso7dstrep was immobilized 17 ; on the other hand, the K D of Sso7dhFc for hFc as determined in experiments where hFc is immobilized is 400 nM 19 .…”

“…The Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus has been used as a versatile scaffold for generation of binding proteins to a wide spectrum of targets, and in the context of several different applications including affinity-based bioseparation and biosensing 17–26 . Further, we show that yeast surface display libraries of mutant proteins containing F2A peptide fusions can be screened to isolate binders with higher affinity.…”

Applications of Yeast Surface Display for Protein Engineering

“…Typically, the amino acid sequence of a contiguous solvent exposed region of the scaffold is randomized to generate a “naïve” library, and the library is displayed on the surface of yeast and screened for binding to a target protein using FACS 47,48 . A number of novel ligands have been engineered using this strategy, including: cysteine knot peptides (knottins) that bind to various integrins with K D values in the picomolar to nanomolar range 47,49–53 , or that inhibit human matriptase-1 with picomolar to nanomolar inhibition constants (K i ) 54 ; human fibronectin 10 th type III domain scaffold 55 variants that bind to a variety of protein targets 18,56,57 , such as lysozyme, with K D values in the nanomolar to picomolar range 29,32 ; green fluorescent protein variants that bind streptavidin-phycoerythrin, biotin-phycoerythrin, glyceraldehyde 3-phosphate dehydrogenase, and a neurotrophin receptor with K D values of 70 nM, 190 nM, 18 nM, and 3.2 nM, respectively 58 ; human kringle domain variants that bind death receptor 4 (DR4), DR5, or TNF-α with K D values of 680 nM, 172 nM, and 29 nM, respectively 59 ; and Sso7d protein variants from the hyperthermophilic archaeon Sulfolobus solfataricus that bind fluorescein, a peptide fragment from β-catenin, hen egg lysozyme, streptavidin, and chicken and mouse immunoglobulins with K D values in the nanomolar to micromolar range 60 .…”