Reproducible enrichment of extracellular heat shock proteins from blood serum using monomeric avidin

Abstract

Extracellular heat shock proteins (eHsps) in blood circulation have been associated with various diseases, including cancer. However, the lack of methods to enrich eHsps from serum samples has hampered the characterization of eHsps. This Letter presents our serendipitous finding that the monomeric avidin resin can serve as an affinity resin to enrich eHsps from blood serum. Biochemical mechanism of this eHsp enrichment as well as implications in biomarker discovery is discussed.

Figure 1

Selective protein enrichment by monomeric avidin. Jurkat cytosolic lysate was incubated with one of the avidin affinity resins. After washing, bound proteins were separated by SDS-PAGE and visualized with Coomassie blue stain. Lane 1: Jurkat cytosolic lysate (no avidin resin treatment); lane 2: Proteins that bound to monomeric avidin resin; lane 3 Proteins that bound to tetrameric avidin resin (no visible bands were observed).

Figure 2

Breakage of tetrameric avidin results in the exposure of interior surfaces. (a) Tetrameric streptavidin (PDB 3MG5). Avidin-avidin interfaces, which are mostly composed of hydrophobic residues, are shown in red, whereas the external hydrophilic surfaces are shown in blue. (b) Monomeric avidin exposes the internal hydrophobic surfaces which serve as the bait for Hsps. (c) Schematic diagram of monomeric avidin resin preparation, in which immobilized tetrameric avidin is chemically denatured and then renatured. A covalent bond between an avidin protein and resin is shown as a purple line. Only covalently attached avidin protein remains on the resin after renaturation.

Figure 3

Affinity purification of serum proteins using monomeric avidin resin. Serum samples from a healthy donor (normal) and a donor with colon cancer (cancer) were incubated with monomeric avidin resin. Proteins bound to the resin were separated by SDS PAGE and visualized by (a) Coomassie-blue staining and (b) silver staining. A notable difference between the two was a silver-stained band around 55–60 kDa (red arrow head)

Figure 4

Expression levels of Hsp60 in serum samples using anti-human Hsp60 antibody. (a) Western blot of serum samples. (b) Quantification of the labeling signals for normal and cancer serum samples. Results are the average values ± standard deviations for four independent experiments (p < 0.05).