Kimberly Trabbic-Carlson

Protein Purification by Fusion with an Environmentally Responsive Elastin‐Like Polypeptide: Effect of Polypeptide Length on the Purification of Thioredoxin

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 ∼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 ∼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.

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion.

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble–insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4‐h culture with chemical induction of the plasmid‐borne fusion protein gene and a 24‐h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24‐h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4‐h–induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in ∼75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high‐throughput, microscale purification; thus, potentially impacting both high‐throughput protein expression and purification for proteomics and large scale, cost‐effective industrial bioprocessing of pharmaceutically relevant proteins.

Ultra-High Expression of a Thermally Responsive Recombinant Fusion Protein in E. coli

Elastin‐like polypeptides (ELPs) are recombinant peptide‐based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient‐rich medium. Compared to commonly used Luria‐Bertani medium, the protein yield was improved by 36‐fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8‐fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP‐fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.

Fusion order controls expression level and activity of elastin-like polypeptide fusion proteins

We have previously developed a method to purify recombinant proteins, termed inverse transition cycling (ITC) that eliminates the need for column chromatography. ITC exploits the inverse solubility phase transition of an elastin‐like polypeptide (ELP) that is fused to a protein of interest. In ITC, a recombinant ELP fusion protein is cycled through its phase transition, resulting in separation of the ELP fusion protein from other Escherichia coli contaminants. Herein, we examine the role of the position of the ELP in the fusion protein on the expression levels and yields of purified protein for four recombinant ELP fusion proteins. Placing the ELP at the C‐terminus of the target protein (protein‐ELP) results in a higher expression level for the four ELP fusion proteins, which also translates to a greater yield of purified protein. The position of the fusion protein also has a significant impact on its specific activity, as ELP‐protein constructs have a lower specific activity than protein‐ELP constructs for three out of the four proteins. Our results show no difference in mRNA levels between protein‐ELP and ELP‐protein fusion constructs. Instead, we suggest two possible explanations for these results: first, the translational efficiency of mRNA may differ between the fusion protein in the two orientations and second, the lower level of protein expression and lower specific activity is consistent with a scenario that placement of the ELP at the N‐terminus of the fusion protein increases the fraction of misfolded, and less active conformers, which are also preferentially degraded compared to fusion proteins in which the ELP is present at the C‐terminal end of the protein.

Surface plasmon optical study of the interfacial phase transition of elastinlike polypeptide grafted on gold

The conformational changes in elastinlike polypeptides (ELPs) grafted to a solid/solution interface via different architectures were studied using surface plasmon resonance spectroscopy and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). SPFS provides a simple and convenient optical method to study the influence of the grafting method and the graft density on the conformational changes in ELPs at the solid-solution interface as a function of environmental variables. A typical response of the ELP, consistent with its stimuli responsiveness, was a gradual collapse upon increasing the ionic strength; this effect was inversely correlated with the surface graft density of the ELP.