Rapid method for measuring ScFv thermal stability by yeast surface display.
We have characterized a simplified method to determine the relative thermal stability of single-chain antibodies by following the irreversible denaturation of scFv fusions on the surface of yeast by flow cytometry. The method was highly reproducible and correlated well with other methods used to monitor thermal denaturation of the soluble proteins. We found a range of thermal stabilities for wild-type single-chain antibodies with half-maximum denaturation temperatures between 43 and 61 degrees C. The ability to quantitate thermal stability of antibodies or other proteins that are immobilized on the surface of yeast allows rapid comparisons of primary structural information with stability. Thermal denaturation could be a useful parameter to consider in the choice of scFv fragments for various applications.
Orr
Brent A
BA
Department of Biochemistry, University of Illinois, 600 S. Matthews Ave., Urbana, Illinois 61801, USA.
Carr
Lori M
LM
Wittrup
K Dane
KD
Roy
Edward J
EJ
Kranz
David M
DM
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism; D004355:Drug Stability; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods; D006358:Hot Temperature; D007128:Immunoglobulin Fragments / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D044366:Transition Temperature
D007128:Immunoglobulin Fragments; C055966:immunoglobulin Fv