A novel system for stable, high-level expression from the T7 promoter

BACKGROUND: The most widespread, efficient prokaryotic protein-producing system is one where the T7 phage polymerase recognizes the T7 phage promoter (T7 p/p system). Unfortunately, in this system, target protein expression gradually declines and is often undetectable following 3 to 5 subcultures. Although a number of studies have attempted to stabilize the expression levels of the T7 p/p system, none has resolved the problem adequately and thus precludes the use of this system for the production of recombinant proteins on a large scale. RESULTS: We created an expression cassette enabling stable, high-level expression in the T7p/p system. The cassette was tested with two different vector backbones and two target proteins. In all experiments, the expression system using the new cassette exhibited high and stable protein expression levels when compared to the traditional system. CONCLUSIONS: Herein, we describe a universal expression cassette that enables high-level, stable target protein expression in T7 RNA polymerase-based expression systems. We also present the successful use of this cassette as a novel expression platform and demonstrate its ability to overcome the main deficiency of the T7 p/p system. Thus, we provide a method for using the T7 p/p system on an industrial scale

(E)-3,3′,4,4′,7,7′,8,8′-Octa­methyl-2H,2′H-1,1′-bi(cyclo­penta­[fg]acenaphthyl­enyl­idene)-2,2′,5,5′,6,6′-hexa­one dichloro­methane monosolvate

The title compound, C36H24O6·CH2Cl2, is a dimer of two essentially planar (r.m.s., deviations of fitted plane of 14 pyracene C atoms = 0.0539 and 0.0543 Å) tetra­cyclic pyracene frameworks (each with four methyl groups and three carbonyl groups on the peripheral carbon atoms) twisted along a central C=C bond with an angle of 50.78 (3)° at 90 K. There are notably long Csp 2—Csp 2 bonds associated with the carbonyl groups, the longest being 1.601 (3) Å between two carbonyl C atoms. There are also intermolecular carbonyl⋯carbonyl interactions of both parallel and antiparallel types, with C⋯O distances in the range 3.041 (3) to 3.431 (2) Å. This compound is of inter­est with respect to the synthesis of fullerene fragments, such as corannulene and semibuckminsterfullerene derivatives (or ‘buckybowls’), and is a side product of the previously reported oxidation reaction. Structural details, such as planarity analysis of fused rings, out-of-plane deviation of substituents, inter­molecular inter­actions, and longer than typical bond lengths, will be discussed as well as comparisons to structurally related compounds

Expression of yeast deubiquitination enzyme UBP1 analogues in E. coli

BACKGROUND: It has been shown that proteins fused to ubiquitin undergo greater expression in E. coli and are easier to purify and renaturate than nonhybrid foreign proteins. However, there is no commercial source of large quantities of specific deubiquitinating proteases. This is the reason why hybrid proteins containing ubiquitin at their N-end cannot be used in large scale biotechnological processes. RESULTS AND CONCLUSION: We have described the synthesis of the yeast deubiquination enzyme UBP1 muteins in E. coli. We have shown that an efficient overproduction of the enzyme in E. coli may be achieved after the introduction of several changes in the nucleotide sequence encoding UBP1. One of the conditions of an effective synthesis of the UBP1 muteins is the removal of the 5'-end sequence encoding the transmembrane region of the enzyme. The obtained variants of the enzyme may be successfully used for processing large amounts of hybrid proteins comprising ubiquitin or tagged ubiquitin at their N-ends

A 2:1 receptor/C60 complex as a nanosized universal joint

Buckycatcher II, a C51 H24 hydrocarbon with two corannulene pincers on a dibenzonorbornadiene tether, exhibits an affinity toward C60 in organic solvents that is dramatically higher than the original buckycatcher C60 H28 and other corannulene-based molecular receptors for fullerenes. In addition to the formation of an usual 1:1 C60 @catcher inclusion complex, a trimeric C60 @(catcher)2 assembly is detected in solutions and in the solid state. X-ray structure determination reveals a remarkable universal joint solvent-free crystal arrangement of the trimer, with a single fullerene cage wrapped by four corannulene subunits of two cooperating catcher receptors

Bis-corannulene Receptors for Fullerenes Based on Klärner\u27s Tethers: Reaching the Affinity Limits

Bis-corannulene receptors 4 and 5 with Klärner\u27s tethers prepared by the Diels-Alder cycloaddition form inclusion complexes with C60 and C70, as evidenced by (1)H NMR titration. While 4 exhibits affinity toward fullerenes comparable to the previously reported corannulene-based receptors, 5 exceeds the performance of the former systems by ca. 2 orders of magnitude and, in addition, shows an enhanced preference for C70 over C60. The X-ray crystal structure of C60@5 and DFT calculations indicate that the tether in 5 not only preorganizes the pincers into a proper topology of the host but also contributes to the dispersion-based binding with the fullerene guests