Simplified protein purification using an autoprocessing, inducible enzyme tag

Abstract

The development of affinity tags has greatly simplified protein purification procedures. A variety of affinity tags are now available to improve expression, solubility, and/or tag removal. In this chapter, we describe a method for purifying recombinant proteins expressed in Escherichia coli that uses a highly specific, inducible, C-terminal autoprocessing protease tag. This method streamlines affinity purification, cleavage, and tag separation into a one-step purification procedure, avoiding the need to remove fusion tags from target proteins with exogenous proteases. In addition to accelerating protein purification, we show that this method can enhance the expression, stability, and solubility of select proteins.

Figure 1

CPD autoprocessing purification system. (a) Schematic of purification scheme as described in text. (b) Schematic of CPD fusion protein and resulting protein post-CPD autoprocessing. (c) Schematic of pET-based cloning vector.

Figure 2

Purification of recombinant proteins using the CPD fusion tag. SDS-PAGE analysis CPD fusion tag-purified proteins using Coomassie staining. (a) Clostridium difficile CspBA (CD2246). When expressed as a C-terminally His₆-tagged protein in E. coli, CspBA exhibits poor solubility (A. Shen, unpublished observations); when expressed as a CPD-His₆-tagged protein in E. coli, CspBA can be purified in a soluble form. (b) mouse macrophage metalloelastin (MMP-12). MMP-12 is insoluble as a His_6- tagged protein in E. coli ([10]). When fused to the CPD, active, untagged MMP-12 can be readily purified from E. coli ([3]). ** indicates the autoprocessed, active form of MMP-12. (c) E. coli biotin ligase BirA. His₆-tgged proteins were bound to Ni-NTA resin then incubated with 50 μM InsP₆ for 1 hr at room temperature; the resin was washed 2-3 times, followed by elution of Ni²⁺-bound proteins by 200 mM imidazole. +, IPTG- induced culture; Pre, imidazole elution prior to InsP₆-mediated cleavage; CL, cleared lysate; SN; supernatant fraction resulting from InsP₆-induced autoprocessing by the CPD.