A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat

Abstract

Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis.

Keywords: BioID; BirA; Protein purification; Proximal biotinylation; Streptavidin; α-Catenin.

Fig. 1

Characterization of cell line and lysis condition. (A) Schematic of proximal biotinylation and GFP-BirA* tagged α-catenin. (B) Western blot analysis of wild type (wt) and BirA*-α-catenin (α) cell line expressing chimeric α-catenin tagged with GFP and BirA*. Western blots were analyzed using α-catenin (top), GFP (middle), and tubulin (bottom) antibodies. (C) Streptavidin Western blot of soluble lysate (S) and insoluble pellet (I) fraction of cell lysate from three different lysis buffers (1, 2, 3 – see Materials and Methods for the buffer compositions). * denotes GFP-BirA*-α-catenin protein band. (D) Streptavidin Western blot of soluble and insoluble fraction of cell lysate from lysis buffer 2 at varying detergent concentrations. (E) Streptavidin Western blot of the lysate, supernatant (supe), eluted biotinylated proteins using excess biotin and heat (initial, bio), and final 4× sample buffer/heat elution (final, 4×) using three different lysis buffers (1, 2, 3).

Fig. 2

Elution efficiency depends on detergent concentration in lysis buffer. (A) Effect of SDS and IGEPAL-CA630 on elution efficiency. Streptavidin Western blot of eluted biotinylated proteins of cultured cells incubated with and without biotin and lysed in buffer 2 containing different detergent compositions. (B) Streptavidin Western blot of lysed cells incubated with and without biotin in lysis buffers 2 of different SDS concentration. The blot shows lysate, eluted biotinylated protein using excess biotin and heat (initial, bio), and final 4× sample buffer/heat elution (final, 4×). (C) Streptavidin blot (left) and quantification (right) of cells incubated with biotin, lysed in lysis buffer 2 with various IGEPAL-CA630 concentrations. Quantification shows the eluted protein with excess biotin/heat (initial, bio) and 4× sample buffer/heat elution (final, 4×), and their sum as a percentage of the bound proteins (calculated based on the difference between lysate and supernatant protein concentrations).

Fig. 3

Optimization of biotinylated elution condition. (A) Proteins eluted (initial) from streptavidin conjugated beads using 0 mM biotin, 25 mM biotin or 4× sample buffer, with or without heating were analyzed with a streptavidin Western blot along with the lysate and supernatant (supe). The remaining bound proteins were eluted with subsequent 4× sample buffer elution (final, 4×). (B) Streptavidin Western blots (top) of lysate, supernatant (supe), initial eluted proteins (initial, bio), and subsequent 4× sample buffer elution (final, 4×). Proteins were initially eluted with different concentrations of biotin at 95 °C for 5 minutes. Quantification (bottom) shows the eluted protein (initial, bio), 4× sample buffer elution (final, 4×), and their sum as a percentage of bound proteins resulting from initial elution with different biotin concentration. Bound proteins were calculated based on the difference in the concentrations of biotinylated proteins detected in the supernatant and the lysate.

Fig. 4

Efficacy of different biotin-binding beads and elution of streptavidin. (A) Binding and elution efficiency with different beads. Streptavidin Western blot of cell lysate (lys), eluted proteins with excess biotin/heat (initial, bio) and 4× sample buffer/heat elution (final, 4×) resulting from cell lysate incubation with different beads: Dynabeads MyOne Streptavidin C1 (C1), Dynabeads M280 Streptavidin (280), and SpeedBead Magnetic Neutravidin Coated Particles (N). (B) Silver stained SDS-PAGE of eluted biotinylated proteins from Dynabead C1 with 25 mM biotin (initial, bio) and final 4× sample buffer elution (final, 4×) with heat. Cell lysates were prepared from cells incubated with (+) and without (-) added biotin in the media. Purified streptavidin (SA) were run on either side of the gel as a size marker for streptavidin.