Browsing by Author "Winslow, Peter"

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    Cloning of MBP-free Protein Variants Using Gibson Assembly
    (2023) Malisetti, Nithya, S; Soltau, Alexander, D; Winslow, Peter; Seelig, Burckhard
    High throughput screening to select for ATP/GTP binding proteins from primordial-like proteins yielded approximately 20 variants with high binding ability. These protein variants with polyhistidine (His) and Maltose Binding Protein (MBP) tags were expressed in Eschericia coli. Tags were added to aid in purification and solubility, but large size of tags caused issues with protein crystallization. Gibson Assembly was explored as a method to successfully clone protein variants without an MBP tag. TEV protease cleavage of His and MBP tagged proteins was also explored to separate MBP from the free protein variants for characterization.
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    Did Early Functional Proteins Use Metal Ions to Bind ATP?
    (2025-01-31) Soltau, Alexander D.; Blascyk, Eli; Winslow, Peter; Seelig, Burckhard
    In early life forms, the first proteins were likely composed of a limited set of amino acid building blocks. Previous work in the Seelig lab has produced random libraries of 85 amino acid-long proteins with ATP binding affinity, made from varying subsets of the 20 modern amino acids. By characterizing these proteins’ binding mechanisms and properties, we hope to gain insight into the nature of early functional proteins. Here, we took initial steps to analyze the metal ion affinity of one such protein composed of five types of amino acids. Further analysis will confirm the dependence of ATP binding on metal ions and extend these investigations to other selected protein variants.