In this Phase I proposal, we plan to develop two technologies to dramatically increase the expression and crystallization of properly folded recombinant membrane proteins from mammalian cell culture. The most common methods to produce high yields of recombinant proteins at low cost rely on bacterial, yeast, or insect-cell expression. However, the proteins produced in these systems are very difficult to purify in native form, and they lack the mammalian glycosylation patterns that are often necessary for proper folding and native activity. We plan to develop a novel protein fusion partner that enables mammalian expression of proteins that are otherwise nearly impossible to produce. A second technology will add an internal motif to greatly enhance structural stability and facilitate crystallization of the target membrane protein. Together, these technologies have the potential to improve protein yields in mammalian cell culture by over 20-fold and to allow crystallization of proteins that are currently impossible to study.
Public Health Relevance Statement: Public Health Relevance: In this proposal, we plan to develop technologies to vastly improve the production of properly folded mammalian membrane proteins, which are critical in many human diseases. This information will allow accurate characterization of the structure and activity of these proteins, and it is essential for rational design of drugs to target them.
Project Terms: Adrenergic Receptor; base; Cell Culture System; Cell Culture Techniques; Cells; Chimeric Proteins; cost; Crystallization; Data; Detection; Detergents; Drug Design; Escherichia coli; Exhibits; expression vector; Fluorescence; G-Protein-Coupled Receptors; Generations; glycosylation; Goals; High temperature of physical object; Human; human disease; improved; Insecta; Integral Membrane Protein; Mammalian Cell; Measures; Membrane Proteins; Methods; novel; overexpression; Pattern; Peptides; Phase; Post-Translational Protein Processing; Process; Production; protein expression; protein purification; Proteins; Proxen; public health relevance; receptor; Recombinant Proteins; Recombinants; Relative (related person); screening; Solubility; structural biology; Structure; System; Technology; therapeutic development; thermostability; Time; tool; trafficking; Variant; Viral; Work; Yeasts
