- Meyer, D.E. and Trabbic-Carlson, K. and Chilkoti, A., Protein purification by fusion with an environmentally responsive elastin-like polypeptide: Effect of polypeptide length on the purification of thioredoxin,
Biotechnology Progress, vol. 17 no. 4
pp. 720 - 728 [bp010049o] .
(last updated on 2007/04/12)
Elastin-like polypeptides (ELPs) undergo a reversible, soluble-to-insoluble phase transition in aqueous solution upon heating through a characteristic transition temperature (Tt). Incorporating a terminal ELP expression tag into the gene of a protein of interest allows ELP fusion proteins to be purified from cell lysate by cycles of environmentally triggered aggregation, separation from solution by centrifugation, and resolubilization in buffer. In this study, we examine the effect of ELP length on the expression and purification of a thioredoxin-ELP fusion protein and show that reducing the size of the ELP tag from 36 to 9 kDa increases the expression yield of thioredoxin by 4-fold, to a level comparable to that of free thioredoxin expressed without an ELP tag, while still allowing efficient purification. However, truncation of the ELP tag also results in a more complex transition behavior than is observed with larger tags. For both the 36 kDa and the 9 kDa ELP tag fused to thioredoxin, dynamic light scattering showed that large aggregates with hydrodynamic radii of [similar to] 2 μm form as the temperature is raised to above the Tt. These aggregates persist at all temperatures above the Tt for the thioredoxin fusion with the 36 kDa ELP tag. With the 9 kDa tag, however, smaller particles with hydrodynamic radii of [similar to] 12 nm begin to form at the expense of the larger, micron-size aggregates as the temperature is further raised above the Tt. Because only large aggregates can be effectively retrieved by centrifugation, efficient purification of fusion proteins with short ELP tags requires selection of solution conditions that favor the formation of the micron-size aggregates. Despite this additional complexity, our results show that the ELP tag can be successfully truncated to enhance the yield of a target protein without compromising its purification.
Polypeptides;Phase transitions;Solutions;Superconducting transition temperature;Genes;Proteins;Cells;Centrifugation;Purification;Aggregates;