Direct Measurement of the Kinetics of CBM9 Fusion-Tag Bioprocessing Using Luminescence Resonance Energy Transfer

摘要

The economics of affinity-tagging technologies,particularly at preparative scales,depends in part on the cost and efficiency of the bioprocessing step used to remove the affinity tag and obtain the final purified product (Lowe et al.,J Biochem Biophys Methods. 2001 ;49:561-574). When CBM9,the family 9 cellulose binding module from Thermotoga maritima,serves as the affinity tag,the overall efficiency of tag removal is a function of the choice of processing enzyme and the local structure of the cleavage site,most notably the linker sequence flanking the bioprocessing recognition site on the tag side. A novel spectroscopic method is reported and used to rapidly and accurately measure CBM9 fusion-tag bioprocessing kinetics and their dependence on the choice of linker sequence. The assay monitors energy transfer between a lanthanide-based donor bound to the CBM9 tag and an acceptor fluorophore presented on the target protein or peptide. Enzyme-catalyzed cleavage of the fusion tag terminates this resonance energy transfer,resulting in a change in fluorescence intensity that can be monitored to quantify substrate concentration over time. The assay is simple,fast and accurate,providing k_(cat)/K_M values that contain standard errors of less than 3%. As a result,both substantial and subtle differences in bioprocessing kinetics can be measured and used to guide bioproduct design.