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This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection(More)
PURPOSE This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeast Saccharomyces cerevisiae. METHODS The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene(More)
Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae(More)
UNLABELLED Integrin αvβ6 is a cell surface receptor minimally expressed by healthy tissue but elevated in lung, colon, skin, ovarian, cervical, and pancreatic cancers. A molecular PET agent for integrin αvβ6 could provide significant clinical utility by facilitating both cancer staging and treatment monitoring to more rapidly identify an effective(More)
The 10th type III domain of human fibronectin (Fn3) has been validated as an effective scaffold for molecular recognition. In the current work, it was desired to improve the robustness of selection of stable, high-affinity Fn3 domains. A yeast surface display library of Fn3 was created in which three solvent-exposed loops were diversified in terms of amino(More)
The rugged protein sequence-function landscape complicates efforts, both in nature and in the laboratory, to evolve protein function. Protein library diversification must strike a balance between sufficient variegation to thoroughly sample alternative functionality versus the probability of mutant destabilization below an expressible threshold. In this(More)
Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two(More)
The development of improved methods for early detection and characterization of cancer presents a major clinical challenge. One approach that has shown excellent potential in preclinical and clinical evaluation is molecular imaging with small-scaffold, non-antibody based, engineered proteins. These novel diagnostic agents produce high contrast images due to(More)
The design of combinatorial libraries for molecular recognition requires extensive diversity to provide high affinity binding to myriad epitopes while maintaining a high degree of functionality to enable inclusion of binders in the limited screenable library size. In the current work, we directly compare minimal and maximal amino acid diversity libraries in(More)
Protein engineering relies on the selective capture of members of a protein library with desired properties. Yeast surface display technology routinely enables as much as million-fold improvements in binding affinity by alternating rounds of diversification and flow cytometry-based selection. However, flow cytometry is not well suited for isolating de novo(More)