![]() Without the application of any affinity maturation strategy, the binding proteins isolated had equilibrium dissociation constants in the nanomolar to micromolar range. Binding proteins for a diverse set of model targets could be isolated from this library our chosen targets included a small organic molecule (fluorescein), a 12 amino acid peptide fragment from the C-terminus of β-catenin, the model proteins hen egg lysozyme and streptavidin, and immunoglobulins from chicken and mouse. We generated a library of 10⁸ Sso7d mutants by randomizing 10 amino acid residues on the DNA-binding surface of Sso7d, using yeast surface display. Sso7d is a small (∼7 kDa, 63 amino acids) DNA-binding protein that lacks cysteine residues and has a melting temperature of nearly 100 ☌. We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Because of their favorable properties, such as ease of recombinant expression, and high thermal, chemical and pH stability, Sso7d-derived binding proteins will have wide applicability in several areas of biotechnology and medicine. They are also resistant to chemical denaturation by guanidine hydrochloride and retain their secondary structure after extended incubation at extreme pH values. Despite extensive mutagenesis, Sso7d mutants have high thermal stability five of six mutants analyzed have melting temperatures >89 ☌. Mutant proteins based on Sso7d were expressed at high yields in the Escherichia coli cytoplasm. Further, Sso7d-derived binding proteins could discriminate between closely related immunoglobulins. ![]() Hopefully, this review will attract the interest of researchers in various fields and empower the development of biocatalysis, biomedicine, environmental remediation, therapy, and diagnosis.We have shown that highly stable binding proteins for a wide spectrum of targets can be generated through mutagenesis of the Sso7d protein from the hyperthermophilic archaeon Sulfolobus solfataricus. Finally, the future development of bioscaffolds for enzyme assembly is also discussed. ![]() In terms of their unique biocompatibility and regenerability, this review mainly covers the current advances in the research and application of non-living and living bioscaffolds with focus on engineering strategies for enzyme assembly. Enzymes assembled onto non-living bioscaffolds are based on single or complex components, while enzymes assembled onto living bioscaffolds are based on living bodies. In view of the unique biocompatibility and regenerability of bioscaffolds, they can be classified into non-living (polysaccharide, nucleic acid, and protein) and living (virus, bacteria, fungi, spore, and biofilm) bioscaffolds, which can fully satisfy these two unique properties, respectively. The regenerability can enable the engineered biocatalysts regenerate through simple self-proliferation without complex re-modification, which is attractive for continuous biocatalytic processes. Biocompatibility makes bioscaffolds more suitable for safe and green production, especially in food processing, production of bioactive agents, and diagnosis. With the demand for green, safe, and continuous biocatalysis, bioscaffolds, compared with synthetic scaffolds, have become a desirable candidate for constructing enzyme assemblages because of their biocompatibility and regenerability.
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