Escherichia coli is the organism of choice for the production of recombinant proteins. Its use as a cell factory is well-established and it has become the most popular expression platform. For this reason, there are many molecular tools and protocols at hand for the high-level production of recombinant proteins, such as a vast catalog of expression plasmids, a great number of engineered strains and many cultivation strategies. We review the different approaches for the synthesis of recombinant proteins in E. coli and discuss recent progress in this ever-growing field.

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Recombinant protein expression in Escherichia coli: advances and challenges.

Biopolyesters polyhydroxyalkanoates (PHA) produced by many bacteria have been investigated by microbiologists, molecular biologists, biochemists, chemical engineers, chemists, polymer experts and medical researchers. PHA applications as bioplastics, fine chemicals, implant biomaterials, medicines and biofuels have been developed and are covered in this critical review. Companies have been established or involved in PHA related R&D as well as large scale production. Recently, bacterial PHA synthesis has been found to be useful for improving robustness of industrial microorganisms and regulating bacterial metabolism, leading to yield improvement on some fermentation products. In addition, amphiphilic proteins related to PHA synthesis including PhaP, PhaZ or PhaC have been found to be useful for achieving protein purification and even specific drug targeting. It has become clear that PHA and its related technologies are forming an industrial value chain ranging from fermentation, materials, energy to medical fields (142 references).

A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry

Bacteria can synthesize a wide range of biopolymers that serve diverse biological functions and have material properties suitable for numerous industrial and medical applications. A better understanding of the fundamental processes involved in polymer biosynthesis and the regulation of these processes has created the foundation for metabolic- and protein-engineering approaches to improve economic-production efficiency and to produce tailor-made polymers with highly applicable material properties. Here, I summarize the key aspects of bacterial biopolymer production and highlight how a better understanding of polymer biosynthesis and material properties can lead to increased use of bacterial biopolymers as valuable renewable products.

Bacterial polymers: biosynthesis, modifications and applications

http://wolfson.huji.ac.il/purification/PDF/Literature/Arnau2006.pdf

Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins.

Biophysical techniques such as fluorescence spectroscopy and nuclear magnetic resonance (NMR) spectroscopy provide a window into the inner workings of proteins. These approaches make use of probes that can either be naturally present within the protein or introduced through a labeling procedure. In general, the more control one has over the type, location and number of probes in a protein, then the more information one can extract from a given biophysical analysis. Recently, two related approaches have emerged that allow proteins to be labeled with a broad range of physical probes. Expressed protein ligation (EPL) and protein trans-splicing (PTS) are both intein-based approaches that permit the assembly of a protein from smaller synthetic and/or recombinant pieces. Here we provide some guidelines for the use of EPL and PTS, and highlight how the dovetailing of these new protein chemistry methods with standard biophysical techniques has improved our ability to interrogate protein function, structure and folding.

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Protein ligation: an enabling technology for the biophysical analysis of proteins

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.

Simple bioseparations using self-cleaving elastin-like polypeptide tags

This work combines two well-established technologies to generate a breakthrough in protein production and purification. The first is the production of polyhydroxybutyrate (PHB) granules in engineered strains of Escherichia coli. The second is a recently developed group of self-cleaving affinity tags based on protein splicing elements known as inteins. By combining these technologies with a PHB-specific binding protein, a self-contained protein expression and purification system has been developed. In this system, the PHB-binding protein effectively acts as an affinity tag for desired product proteins. The tagged product proteins are expressed in E. coli strains that also produce intracellular PHB granules, where they bind to the granules via the PHB-binding tag. The granules and attached proteins can then be easily recovered following cell lysis by simple mechanical means. Once purified, the product protein is self-cleaved from the granules and released into solution in a substantially purified form. This system has been successfully used at laboratory scale to purify several active test proteins at reasonable yield. By allowing the bacterial cells to effectively produce both the affinity resin and tagged target protein, the cost associated with the purification of recombinant proteins could be greatly reduced. It is expected that this combination of improved economics and simplicity will constitute a significant breakthrough in both large-scale production of purified proteins and enzymes and high-throughput proteomics studies of peptide libraries.

/pdf/novel-and-economical-purification-of-recombinant-proteins-52umng6p7w.pdf

Novel and economical purification of recombinant proteins: Intein‐mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association

The development of self-cleaving fusion-tag technology has greatly simplified the purification of recombinant proteins at laboratory scale. The self-cleaving capability of these tags has recently been combined with additional purification tags to generate novel and convenient protein purification methods at a variety of scales. In this review, we describe some of these methods, and provide a rudimentary economic analysis of hypothetical large-scale applications. This work is expected to provide a rough outline for the evaluation of these methods for large-scale bioprocessing of a variety of products.

https://link.springer.com/content/pdf/10.1186%2F1475-2859-4-32.pdf

Inteins and affinity resin substitutes for protein purification and scale up

Towards the elimination of chromatography in protein purification: Expressing proteins engineered to purify themselves

Purification of recombinant proteins is performed by expressing in a host cell a fusion protein comprising: (a) a product protein domain, (b) an intein, and (c) at least one aggregator protein domain, wherein the aggregator protein domain comprises a protein that is capable of specific association with granules of polyhydroxyalkanoate (PHA).

Mahmoud Reza Banki

Papers

Simple bioseparations using self-cleaving elastin-like polypeptide tags

Novel and economical purification of recombinant proteins: Intein‐mediated protein purification using in vivo polyhydroxybutyrate (PHB) matrix association

Inteins and affinity resin substitutes for protein purification and scale up

Towards the elimination of chromatography in protein purification: Expressing proteins engineered to purify themselves

Intein-mediated protein purification using in vivo expression of an aggregator protein