Measuring protein interactions by microchip self-interaction chromatography.
The self-interaction of proteins is of paramount importance in aggregation and crystallization phenomena. Solution conditions leading to a change in the state of aggregation of a protein, whether amorphous or crystalline, have mainly been discovered by the use of trial and error screening of large numbers of solutions. Self-interaction chromatography has the potential to provide a quantitative method for determination of protein self-interactions amenable to high-throughput screening. This paper describes the construction and characterization of a microchip separation system for low-pressure self-interaction chromatography using lysozyme as a model protein. The retention time was analyzed as a function of mobile-phase composition, amount of protein injected, flow rate, and stationary-phase modification. The capacity factors (k') as a function of crystallizing agent concentration are compared with previously published values for the osmotic second virial coefficient (B(22)) obtained by static light scattering, showing the ability of the chip to accurately determine protein-protein interactions. A 500-fold reduction in protein consumption and the possibility of using conventional instrumentation and automation are some of the advantages over currently used methodologies for evaluating protein-protein interactions.
García
Carlos D
CD
Department of Chemistry, Mississippi State University, Mississippi State, Mississippi 39762, USA.
Hadley
DeGail J
DJ
Wilson
W William
WW
Henry
Charles S
CS
D002845:Chromatography / Q000295:instrumentation* / Q000379:methods*; D003460:Crystallization / Q000379:methods*; D019281:Dimerization; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005240:Feasibility Studies; D008904:Miniaturization / Q000379:methods; D009113:Muramidase / Q000032:analysis / Q000737:chemistry; D040081:Protein Array Analysis / Q000295:instrumentation* / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation* / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D011506:Proteins; D009113:Muramidase