User:Kevin Dooley

Protein and Metabolic Engineering

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Kevin Dooley

• Kevin Dooley
• Columbia University
• 500 W120th Street
• Mudd 801
• New York, NY 10027
• kpd2109@columbia.edu

I work in Scott Banta's Protein and Metabolic Engineering Lab at Columbia University.

Education

• 2014, PhD, Chemical Engineering, Columbia University, New York, NY
• 2009, MS, Chemical Engineering, Manhattan College, Bronx, NY
• 2008, BS, Chemical Engineering, Manhattan College, Bronx, NY

Research interests

The majority of my research focuses on engineering a conformationally dynamic peptide isolated from a repeats-in-toxin (RTX) domain. This protein scaffold undergoes a reversible structural rearrangement in response to low millimolar concentrations of calcium from an intrinsically disordered state to a compact β-roll structure. We use this triggered conformational response to mediate a variety of molecular interactions for use in biotechnology systems, outlined below.

• We have have rationally designed a leucine-rich RTX mutant capable of forming intermolecular cross-links for calcium-responsive proteinaceous hydrogel formation. In the absence of calcium, the designed hydrophobic interface is delocalized, thus mitigating the driving force for self-assembly.
  

• RTX proteins are composed of a repeating nonamer calcium binding sequence. Often times for repeating protein scaffolds, consensus design is used to create synthetic peptides based on the amino acid frequency at each position. This can lead to higher levels of stability and recombinant expression. The genetic information can also be optimized for easier cloning and concatenation strategies. In exploring consensus design for the RTX protein, we discovered a synthetic sequence that undergoes a reversible phase change in response to calcium binding. We have used this sequence as a non-chromatographic protein purification tag, similar to the elastin like peptide (ELP) tags. By appending the β-roll tag (BRT) to a protein of interest, we can rapidly and efficiently separate the fusion from cell lysate (~ 10 minutes). We have added a protease site between the BRT and protein of interest allowing for the purification of untagged target by precipitation cycling. The image below shows the calcium specificity of the BRT. Fusions to GFP were prepared and incubated in a variety of ions (CaCl₂, MgCl₂, MnCl₂, NaCl, (NH₄)₂SO₄, respectively). Note the near complete removal of GFP from solution in the first tube treated with calcium.

• We are also currently evaluating the RTX protein as an alternative scaffold for evolving molecular recognition. The calcium-induced structural rearrangement can be used as a switch to turn a designed binding interface "on" and "off" thus creating an allosterically regulated binding domain. We have constructed an RTX library by randomizing 8 solvent exposed positions on the face of the folded β-roll structure and are currently selecting for affinity against several interesting targets using ribosome display technology.

Publications

1. Banta, S Dooley, K, Shur, O. (2013) Replacing antibodies: Engineering new binding proteins. Annual review of biomedical engineering 15: 93-113
2. Shur O, Dooley K, Blenner M, Baltimore M, Banta S. (2013) A designed, phase-changing RTX-based peptide for efficient bioseparations. Biotechniques 54(4): 197-206
3. Dooley K, Kim YH, Lu HD, Tu R, Banta S. (2012) Engineering of an Environmentally Responsive Beta Roll Peptide for Use As a Calcium-Dependent Cross-Linking Domain for Peptide Hydrogel Formation. Biomacromolecules 13: 1758-64
4. Wu J, Park JP, Dooley K, Cropek DM, West AC, Banta S. (2011) Rapid Development of New Protein Biosensors Utilizing Peptides Obtained via Phage Display. PLOS ONE 6: e24948
5. Sahin A, Dooley K, Cropek DM, West AC, Banta S. (2011) A dual enzyme electrochemical assay for the detection of organophosphorus compounds using organophosphorus hydrolase and horseradish peroxidase. Sensors and Actuators B: Chemical 158: 353-60