Disulfide trapping complexes

Coexpression of disulfide-trapped complexes between receptors and chemokines in insect Sf9 cells is similar to coexpression of noncovalent complexes and is described in detail by Gustavsson et al in Chapter 11. Briefly, the PI stocks of the two types of particles (those carrying receptor and those carrying chemokine) are used to coinfect Sf9 cells at a density of 2—2.6 X 10° ceUs/mL. Even if the multiphcity of infection (MOI) has been previously separately optimized for each vector, it frequently needs to be reoptimized in the context of the coexpression experiment, likely because the cell expression machinery gets taxed to a different degree when producing individual components versus the complex (Fig. 5). 

Characterization of Monodispersity and Thermal Stability of Disulfide-Trapped Complexes with SEC and CPM-DSF... 

SEC and CPM-DSF represent the most stringent and specific assays for characterization of disulfide-trapped complexes. This is because even the most clear-cut crossfinks may introduce minor distortions to the native favorable positions and orientations of the receptor and the chemokine in the complex such distortions are detectable by lowered thermal stability or increased aggregation of the purified complexes as compared to an ideal geometry crossfink. 

Figure 11 CPM-DSF characterization of noncovalent and disulfide-trapped complexes of a CXC chemokine and its receptor. (A) Melting curves plotting the increasing fluorescence of the CPM dye as a function of temperature. A biphasic transition is observed for the disulfide-trapped sample, likely due to the presence of both noncovalent and disulfide-trapped subpopulations. (B) First derivative curves of (A) demonstrate the positions of the peaks used to calculate Tm- A double-peak is clearly present for the disulfide-trapped sample. (C) Melting temperatures are calculated from the derivative curves.The disulfide-trapped sample shows two TmS one of which is indistinguishable from the noncovalent sample Tm-...

Disulfide Trapping for Modeling and Structure Determination of ReceptorrChemokine Complexes... 

Table 1 Comparison of Experimental Approaches for Characterization of Disulfide-Trapped ReceptonChemokine Complexes... 

The result of a flow cytometry experiment is a histogram of cell distribution by the total amount of surface-bound chemokine, with or without an unlabeled competitor Hgand. This assay provides an easy way to quickly screen numerous candidate disulfide-trapped construct combinations uHth-out the complexities of sample purification however, the utUity of this method is limited to chemokines that are detectable on the cell surface by a... 

Hagemann, I. S., Miller, D. L., Klco, J. M., Nikiforovich, G. V., Baranski, T. J. (2008). Structure of the complement factor 5a receptor-hgand complex studied by disulfide trapping and molecular modeling. Journal of Biological Chemistry, 283(12), 7763—7775. http // dx.doi.org/10.1074/jbc.M709467200. 

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