Browsing by Author "Seelig, Burckhard"

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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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    TEV Protease Cleavage of MBP and His Affinity Tags in de novo Fusion Proteins
    (2023-07) Soltau, Alexander D.; Malisetti, Nithya; Winslow, Peter J; Seelig, Burckhard
    Polyhistidine (His) and Maltose Binding Protein (MBP) tagged proteins were successfully expressed in Escherichia coli. TEV protease cleavage and MBP-free cloning were pursued as strategies for obtaining pure, MBP-free protein variants for further research. A protocol for TEV cleavage was developed, and methods of subsequent purification or alternative cloning were explored.