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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)
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. The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene dosage. Secreted scFv(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)
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 human fibronectin type III domain ((10)Fn3) is one of several protein scaffolds used to design and select families of proteins that bind with high affinity and specificity to macromolecular targets. To date, the highest affinity (10)Fn3 variants have been selected by mRNA display of libraries generated by randomizing all three(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)
PURPOSE The aim of this article was to evaluate the use of a novel engineered anti-CD20 protein based on the 10 kDa human fibronectin type 3 domain (FN3) and subsequently compare with (64)Cu-rituximab for positron emission tomography (PET) imaging of CD20. EXPERIMENTAL DESIGN The engineered FN3(CD20) and FN3(WT) were produced in Escherichia coli cells at(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)
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)