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.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a member of the PARP enzyme family consisting of PARP-1 and several recently identified novel poly(ADP-ribosylating) enzymes. PARP-1 is an abundant nuclear protein functioning as a DNA nick-sensor enzyme. Upon binding to DNA breaks, activated PARP cleaves NAD(+) into nicotinamide and ADP-ribose and polymerizes the latter onto nuclear acceptor proteins including histones, transcription factors, and PARP itself. Poly(ADP-ribosylation) contributes to DNA repair and to the maintenance of genomic stability. On the other hand, oxidative stress-induced overactivation of PARP consumes NAD(+) and consequently ATP, culminating in cell dysfunction or necrosis. This cellular suicide mechanism has been implicated in the pathomechanism of stroke, myocardial ischemia, diabetes, diabetes-associated cardiovascular dysfunction, shock, traumatic central nervous system injury, arthritis, colitis, allergic encephalomyelitis, and various other forms of inflammation. PARP has also been shown to associate with and regulate the function of several transcription factors. Of special interest is the enhancement by PARP of nuclear factor kappa B-mediated transcription, which plays a central role in the expression of inflammatory cytokines, chemokines, adhesion molecules, and inflammatory mediators. Herein we review the double-edged sword roles of PARP in DNA damage signaling and cell death and summarize the underlying mechanisms of the anti-inflammatory effects of PARP inhibitors. Moreover, we discuss the potential use of PARP inhibitors as anticancer agents, radiosensitizers, and antiviral agents.

The Therapeutic Potential of Poly(ADP-Ribose) Polymerase Inhibitors

http://teachline.ls.huji.ac.il/72682/Publications/Sorensen2005.pdf

Advanced genetic strategies for recombinant protein expression in Escherichia coli.

Using a recently described self-assembly process (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Letters 2002, 2, 853−856), we prepared soluble monodisperse discoidal lipid/protein particles with controlled size and composition, termed Nanodiscs, in which the fragment of dipalmitoylphosphatidylcholine (DPPC) bilayer is surrounded by a helical protein belt. We have customized the size of these particles by changing the length of the amphipathic helical part of this belt, termed membrane scaffold protein (MSP). Herein we describe the design of extended and truncated MSPs, the optimization of self-assembly for each of these proteins, and the structure and composition of the resulting Nanodiscs. We show that the length of the protein helix surrounding the lipid part of a Nanodisc determines the particle diameter, as measured by HPLC and small-angle X-ray scattering (SAXS). Using different scaffold proteins, we obtained Nanodiscs with the average size from 9.5 to 12.8 nm with a very narrow size distributi...

Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size.

Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.

The P1' specificity of tobacco etch virus protease.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2891%2980362-7

József Tözsér

Papers

Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency.

The P1' specificity of tobacco etch virus protease.

Comparison of the HIV‐1 and HIV‐2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag‐Pol polyproteins

Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro.

Beta-lactam compounds as apparently uncompetitive inhibitors of HIV-1 protease