Expression and purification of ELP-intein-tagged target proteins in high cell density E. coli fermentation.

BACKGROUND: Elastin-like polypeptides (ELPs) are useful tools that can be used to non-chromatographically purify proteins. When paired with self-cleaving inteins, they can be used as economical self-cleaving purification tags. However, ELPs and ELP-tagged target proteins have been traditionally expressed using highly enriched media in shake flask cultures, which are generally not amenable to scale-up. RESULTS: In this work, we describe the high cell-density expression of self-cleaving ELP-tagged targets in a supplemented minimal medium at a 2.5 liter fermentation scale, with increased yields and purity compared to traditional shake flask cultures. This demonstration of ELP expression in supplemented minimal media is juxtaposed to previous expression of ELP tags in extract-based rich media. We also describe several sets of fed-batch conditions and their impact on ELP expression and growth medium cost. CONCLUSIONS: By using fed batch E. coli fermentation at high cell density, ELP-intein-tagged proteins can be expressed and purified at high yield with low cost. Further, the impact of media components and fermentation design can significantly impact the overall process cost, particularly at large scale. This work thus demonstrates an important advances in the scale up of self-cleaving ELP tag-mediated processes.

Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag.

A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP alpha subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the beta, beta', and omega subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target.

The potential role of self-cleaving purification tags in commercial-scale processes.

Purification tags are robust tools that can be used to purify a wide selection of target proteins, which makes them attractive candidates for implementation into platform processes. However, tag removal remains an expensive and significant issue that must be resolved before these tags can become widely used. One alternative is self-cleaving purification tags, which can provide the purity and versatility of conventional tags but eliminate the need for proteolytic tag removal. Many of these self-cleaving tags are based on inteins, but other emerging technologies, such as the FrpC and SrtAc proteins, have also been reported. In this review, we cover affinity and non-chromatographic self-cleaving purification tags and their potential industrial applications.

Recombinant protein purification by self-cleaving elastin-like polypeptide fusion tag.

This unit presents a rapid and simple method for the nonchromatographic purification of recombinant proteins expressed in E. coli. This method relies on a thermally responsive elastin-like polypeptide (ELP) tag, where the tagged protein is precipitated using a mild temperature shift. The tag is then induced to self-cleave by a mild pH shift and is subsequently removed by a final thermal precipitation. The result is a purified native protein target, without the requirement for affinity apparatus or protease removal of the tag. This protocol describes the required cloning methods to insert a given target into the expression vector, as well as the general method for purifying the resulting expressed protein.

Optimization of ELP-intein mediated protein purification by salt substitution.

In this paper we discuss improvements to our previously reported ELP-intein purification system described by Banki et al. [M.R. Banki, L. Feng, D.W. Wood, Simple bioseparations using self-cleaving elastin-like polypeptide tags, Nat. Methods 2 (2005) 659-661; W.Y. Wu, C. Mee, F. Califano, R. Banki, D.W. Wood, Recombinant protein purification by self-cleaving aggregation tag, Nat. Protoc. 1 (2006) 2257-2262]. This method is based on the selective and reversible precipitation of ELP-tagged target proteins by gentle heating in the presence of high concentrations of sodium chloride. A critical aspect of this system is that the ELP tag is induced to self-cleave by a mild pH shift after purification. An examination of the Hofmeister series of ions suggested that salts other than sodium chloride may be more efficient for ELP precipitation. Specifically, by replacing sodium chloride with ammonium sulfate to induce ELP aggregation, we were able to reduce the required salt concentration by almost 4-fold, and the precipitation steps could be conducted at room temperature instead of 37 degrees C. This results in a cheaper, gentler, and more scaleable purification method. To demonstrate these advantages, green fluorescent protein and beta-lactamase were purified using the newly optimized conditions in side-by-side comparisons to the previous method. The results indicate that both specific activity and yield were improved with the new conditions. These improvements thus significantly increase the attractiveness of this highly general and economical method for recombinant protein purification.