Improved self-cleaving precipitation tags for efficient column free bioseparations.

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

Single-Step Non-Chromatographic Purification of Recombinant Mammalian Proteins Using a Split Intein ELP Tag System.

Glycoprotein therapeutics are currently used by large patient populations and generate significant revenue for the biopharmaceutical industry. These therapeutic proteins are currently purified at industrial scale using individualized processes involving multiple chromatographic steps. In the absence of a viable affinity platform method, the required chromatographic steps are difficult to develop and inevitably lead to significant yield losses. Further, during preclinical development, there is a need for reliable platform technologies capable of performing high-throughput screening for biologic candidates. Although affinity tags can provide a solution to some of these challenges, they require specific affinity resins, and the tag itself can interfere with the target protein characteristics. Fusion protein systems consisting of elastin-like polypeptide (ELP) and self-cleaving split inteins such as Npu DnaE can serve as potential non-chromatographic platform technologies for the single-step purification of tagless glycoproteins expressed in mammalian cells. In this chapter, we demonstrate the use of this technology to obtain highly purified anti-ErbB2 ML39 single-chain variable fragment (scFv) expressed from Expi293F suspension cells.

Mechanistic discoveries and simulation-guided assay optimization of portable hormone biosensors with cell-free protein synthesis.

Nuclear receptors (NRs) influence nearly every system of the body and our lives depend on correct NR signaling. Thus, a key environmental and pharmaceutical quest is to identify and detect chemicals which interact with nuclear hormone receptors, including endocrine disrupting chemicals (EDCs), therapeutic receptor modulators, and natural hormones. Previously reported biosensors of nuclear hormone receptor ligands facilitated rapid detection of NR ligands using cell-free protein synthesis (CFPS). In this work, the advantages of CFPS are further leveraged and combined with kinetic analysis, autoradiography, and western blot to elucidate the molecular mechanism of this biosensor. Additionally, mathematical simulations of enzyme kinetics are used to optimize the biosensor assay, ultimately lengthening its readable window by five-fold and improving sensor signal strength by two-fold. This approach enabled the creation of an on-demand thyroid hormone biosensor with an observable color-change readout. This mathematical and experimental approach provides insight for engineering rapid and field-deployable CFPS biosensors and promises to improve methods for detecting natural hormones, therapeutic receptor modulators, and EDCs.

Recent Advances in the Scaffold Engineering of Protein Binders.

In recent years, extensive attention has been given to the generation of new classes of ligand- specific binding proteins to supplement monoclonal antibodies. A combination of protein engineering and display technologies has been used to manipulate non-human antibodies for humanization and stabilization purposes or even the generation of new binding proteins. Engineered protein scaffolds can now be directed against therapeutic targets to treat cancer and immunological disorders. Although very few of these scaffolds have successfully passed clinical trials, their remarkable properties such as robust folding, high solubility, and small size motivate their employment as a tool for biology and applied science studies. Here, we have focused on the generation of new non-Ig binding proteins and single domain antibody manipulation, with a glimpse of their applications.

Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach.

Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor ß (hERß). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERß (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.

Column-Free Purification Methods for Recombinant Proteins Using Self-Cleaving Aggregating Tags.

Conventional column chromatography processes to purify recombinant proteins are associated with high production costs and slow volumetric throughput at both laboratory and large scale. Non-chromatographic purifications based on selective aggregating tags have the potential to reduce costs with acceptable protein yields. A significant drawback, however, is that current proteolytic approaches for post-purification tag removal after are expensive and non-scalable. To address this problem, we have developed two non-chromatographic purification strategies that use either the elastin-like polypeptide (ELP) tag or the ß-roll tag (BRT17) in combination with an engineered split intein for tag removal. The use of the split intein eliminates premature cleavage during expression and provides controlled cleavage under mild conditions after purification. These self-cleaving aggregating tags were used to efficiently purify ß-lactamase (ß-lac), super-folder green fluorescent protein (sfGFP), streptokinase (SK) and maltose binding protein (MBP), resulting in increased yields compared to previous ELP and BRT17-based methods. Observed yields of purified targets for both systems typically ranged from approximately 200 to 300 micrograms per milliliter of cell culture, while overall recoveries ranged from 10 to 85 percent and were highly dependent on the target protein.

Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

Bacterial biosensors for evaluating potential impacts of estrogenic endocrine disrupting compounds in multiple species.

To study the effects and possible mechanisms of suspected endocrine disrupting compounds (EDCs), a wide variety of assays have been developed. In this work, we generated engineered Escherichia coli biosensor strains that incorporate the ligand-binding domains (LBDs) of the ß-subtype estrogen receptors (ERß) from Solea solea (sole), and Sus scrofa (pig). These strains indicate the presence of ligands for these receptors by changes in growth phenotype, and can differentiate agonist from antagonist and give a rough indication of binding affinity via dose-response curves. The resulting strains were compared with our previously reported Homo sapiens ERß biosensor strain. In initial tests, all three of the strains correctly identified estrogenic test compounds with a high degree of certainly (Z' typically greater than 0.5), including the weakly binding test compound bisphenol A (BPA) (Z' ≈ 0.1-0.3). The modular design of the sensing element in this strain allows quick development of new species-based biosensors by simple LBD swapping, suggesting its use in initial comparative analysis of EDC impacts across multiple species. Interestingly, the growth phenotypes of the biosensor strains indicate similar binding for highly estrogenic control compounds, but suggest differences in ligand binding for more weakly binding EDCs.

Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands.

The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G(4)S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD.

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.

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.

Application of screening methods, shape signatures and engineered biosensors in early drug discovery process.

PURPOSE: In this study, two unreported estrogen antagonists were identified using a combination of computational screening and a simple bacterial estrogen sensor. METHODS: Molecules here presented were initially part of a group obtained from a library of over a half million chemical compounds, using the Shape Signatures method. The structures within this group were then clustered and compared to known antagonists based on their physico-chemical parameters, and possible binding modes of the compounds to the Estrogen Receptor alpha (ER alpha) were analyzed. Finally, thirteen candidate compounds were purchased, and two of them were shown to behave as potential subtype-selective estrogen antagonists using a set of bacterial estrogen biosensors, which included sensors for ER alpha, ER beta, and a negative control thyroid hormone beta biosensor. These activities were then analyzed using an ELISA assay against activated ER alpha in human MCF-7 cell extract. RESULTS: Two new estrogen receptor antagonists were detected using in silico Shape Signatures method with an engineered subtype-selective bacterial estrogen biosensor and commercially available ELISA assay. Additional thyroid biosensor control experiments confirmed no compounds interacted with human thyroid receptor beta. CONCLUSIONS: This work demonstrates an effective combination of computational analysis and simple bacterial screens for rapid identification of potential hormone-like therapeutics.

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.

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags.

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.

Verification of biochemical activity for proteins nanografted on gold surfaces.

We demonstrate that the Atomic Force Microscope (AFM) can be used to immobilize a dicysteine-terminated protein (Maltose Binding Protein, MBP-cys-cys for short) at well-defined locations directly on gold substrates via nanografting and characterize the in situ bioactivity of these proteins within the fabricated nanopatterns. This method exploits the high spatial and orientational control of the protein monolayer assembly allowed by nanografting, combined with the high sensitivity of the AFM for detecting ligand-binding events. The maltose-mediated conformational changes within the MBP have been found to change the AFM-tip-protein interaction, therefore causing the frictional signal to change. Our measurements show that the protein ligand-binding function is maintained upon the immobilization process and is not affected by (a) the addition of the cysteine dipeptide, (b) the spatial confinement associated with nanografting, and (c) the interaction between the protein and the Au substrate. These surface-confined proteins can also be regenerated, and their frictional response is reproducible through several maltose exposure/washing cycles. By measuring the change in the frictional force above the protein nanopatterns as a function of maltose concentration, we determined the dissociation constant for the MBP-cys-cys/maltose system to be kd = (1 +/- 0.04) microM. Our results show that the MBP-cys-cys system provides a very sensitive surface-based, protein nanobiosensor for maltose detection at the attogram level (approximately 100 nM concentration). The implications of our study for the fabrication of molecular-scale biological sensors are discussed at the end of the paper.

Engineered chimeric enzymes as tools for drug discovery: generating reliable bacterial screens for the detection, discovery, and assessment of estrogen receptor modulators.

Engineered protein-based sensors of ligand binding have emerged as attractive tools for the discovery of therapeutic compounds through simple screening systems. We have previously shown that engineered chimeric enzymes, which combine the ligand-binding domains of nuclear hormone receptors with a highly sensitive thymidylate synthase reporter, yield simple sensors that report the presence of hormone-like compounds through changes in bacterial growth. This work describes an optimized estrogen sensor in Escherichia coli with extraordinary reliability in identifying diverse estrogenic compounds and in differentiating between their agonistic/antagonistic pharmacological effects. The ability of this system to assist the discovery of new estrogen-mimicking compounds was validated by screening a small compound library, which led to the identification of two structurally novel estrogen receptor modulators and the accurate prediction of their agonistic/antagonistic biocharacter in human cells. Strong evidence is presented here that the ability of our sensor to detect ligand binding and recognize pharmacologically critical properties arises from allosteric communication between the artificially combined protein domains, where different ligand-induced conformational changes in the receptor are transmitted to the catalytic domain and translated to distinct levels of enzymic efficiency. To the best of our knowledge, this is one of the first examples of an engineered enzyme with the ability to sense multiple receptor conformations and to be either activated or inactivated depending on the nature of the bound effector molecule. Because the proposed mechanism of ligand dependence is not specific to nuclear hormone receptors, we anticipate that our protein engineering strategy will be applicable to the construction of simple sensors for different classes of (therapeutic) binding proteins.

Recombinant protein purification by self-cleaving aggregation tag.

A simple technique is presented for non-chromatographic purification of recombinant proteins expressed in Escherichia coli. This method is based on a reversibly precipitating, self-cleaving purification tag. The tag is made up of two components: an elastin-like polypeptide (ELP), which reversibly self-associates in high-salt buffers at temperatures above 30 degrees C; and an intein, which causes the ELP tag to self-cleave in response to a mild pH shift. Thus, a tripartite ELP-intein-target protein precursor can be purified by cycles of salt addition, heating and centrifugation. Once purified, intein-mediated self-cleavage, followed by precipitation of the cleaved ELP tag, allows easy and effective isolation of the pure, native target protein without the need for chromatographic separations. Recoveries of 50-100 mg of cleaved, native target protein per liter of shake-flask culture have been achieved for over a dozen proteins, typically in 8-24 h depending on specific process parameters.

Rapid detection of subtype-selective nuclear hormone receptor binding with bacterial genetic selection.

Subtype-selective nuclear hormone receptor modulators could potentially allow the development of valuable tissue-specific therapeutics. A simple biosensor that allows subtype-specific nuclear hormone receptor binding to be reflected by the growth phenotype of Escherichia coli cells has been constructed. This system will potentially enable the facile detection or evolution of subtype-selective hormone analogues.

Simple bioseparations using self-cleaving elastin-like polypeptide tags.

We introduce a new method for the purification of recombinant proteins expressed in Escherichia coli using self-cleaving elastin-like polypeptide (ELP) fusion tags without the need for affinity chromatography or proteolytic tag removal. Using this method we obtained high purity, activity and reasonable yields for ten diverse target proteins.