ABSTRACT:We have combined Invitrogen's Gateway 1 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 DI-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 selfcleaving 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 b-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 DI-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.

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 a subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the b, b 0 , and x 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.

A method has been developed that eliminates the need for complex chromatographic apparatus in the purification of recombinant proteins expressed in Escherichia coli. This method is similar to conventional affinity-tag separations, but the affinity resin is replaced by polyhydroxybutyrate (PHB) particles prodced in vivo in the E. coli expression host during protein expression. A PHB-binding protein known as a phasin is genetically fused to the product protein via an engineered pH and temperature dependent self-cleaving intein linker. Thus the phasin-sion acts as a self-cleaving purification tag, with affinity for the co-expressed PHB granules. The PHB particles and tagged target protein are purified by lysing the cells and washing the granules with sequential rounds of centrifugation and resuspension. The native target protein is then released from the bound tag through an intein-mediated self-cleavage reaction, induced by a mild pH shift. A final round of centrifugation removes the granules and associated tag, allowing the purified target to be recovered in the supernatant. This method has been shown to yield 35-40 microg of purified product per milliliter of liquid cell culture and is likely to be applicable to a wide range of expression hosts.

This work demonstrates an effective combination of computational analysis and simple bacterial screens for rapid identification of potential hormone-like therapeutics.

Biomolecular engineering has many applications in the identification of potentially therapeutic compounds. An important class of these compounds is those that bind and modulate the activity of the human nuclear hormone receptors (NHRs). NHRs are typically made up of clearly defined domains with known function, including one that mediates ligand recognition and NHR activation. Engineered systems that include these ligand‐binding domains (LBDs) can be used to identify potential therapeutic ligands that target a given NHR. These methods must couple the binding event to a readily detectable signal, ideally in a high‐throughput format. Recent efforts have delivered a variety of new techniques, including those that involve fusions of LBDs to easily assayed reporter proteins. In some cases these systems allow hormone‐dependent selectable phenotypes to be generated in non‐native hosts, providing potential tools for both isolation and evolution of new therapeutics in vivo. Here we provide an overview and a comparison of many of the available tools in this area, with an emphasis on a novel allosteric hormone‐regulated sensor protein that provides ligand‐dependent phenotypes in the relatively simple background of Escherichia coli bacterial cells.