Bacterial expression

Lyons, R.E., Lesieur, E., Kim, M., Wong, D., Brownlee, A., Pearson, R., and Elvin, C., The development of synthetic genes encoding repetitive resilin-like polypeptides Construct design, bacterial expression and rapid purification. Protein Eng. Design Select., 20(1), 25-32, 2007. 

Besides short ELPS, longer ELPs have also been conjugated to synthetic polymers. In one approach, Cu(I)-catalyzed azide-alkyne cycloaddition click chemistry was applied. For this purpose, ELPs were functionalized with azides or alkynes via incorporation of azidohomoalanine and homopropargyl glycine, respectively, using residue-specific replacement of methionine in ELP via bacterial expression [133]. More recently, an alternative way to site-selectively introduce azides into ELPs was developed. Here, an aqueous diazotransfer reaction was performed directly onto ELP[V5L2G3-90] using imidazole-1-sulfonyl azide [134]. 

One of the best-studied carrier molecules is produced as a primary excretory constituent of the adult male mouse, known from its consistent high concentration as the major urinary protein (MUP). The basic 3-D structure of the protein was initially obtained from a monoclinic crystal of recombinant protein (MUP-I), constructed by induction in a bacterial expression system and purified to homogeneity (Kuser, 1990). A wild type version of MUP finally yielded to NMR analysis a clone of the r-isoform (162 residues) was labelled and compared with the crystal-structure (Lucke et al., 1990). Two views of the molecule... 

Terpe, K. (2006) Overview of bacterial expression systems for heterologous protein production from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 72 (2), 211-222. 

Consequently, the signal fractions of 51 f(r)/51 f(0) can be calculated and compared with simulations. For samples prepared by bacterial expression, correction of the background signals can be carried out in a similar fashion [48, 53, 54], It is noteworthy that spin geometries deviated from linearity, which may occur in the side-chain 13C nuclei such as C , would produce more rapid signal decays and smaller residual signal fraction [55],... 

FIGURE 45-5 Filaments extracted from the brains of patients with dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) or assembled from bacterially expressed human a-synuclein (SYN) were decorated by an anti-a-synuclein antibody. The gold particles conjugated to the second antibody appear as black dots. Scale bar lOOnm. 

New insights into the analysis of hydrophobically post-translational modified proteins could be achieved by the construction of lipidated proteins in a combination of bioorganic synthesis of activated lipopeptides and bacterial expression of the protein backbone (Fig. 19). The physico-chemical properties of such artificial lipoproteins differ substantially from those of the corresponding lipopeptides. The pronounced dominance of the hydrophilic protein moiety (e.g., for the Ras protein 181 amino acids) over a short lipopeptide with one or two hydrophobic modifications provides solubility up to 10 4 mol/1, while the biotinylated or fluorescence labeled lipopeptides exhibit low solubility in aqueous solutions and can be applied in the biophysical experiments only in vesicle integrated form or dissolved in organic solvent. 

Despite the data reviewed here, the work is not finished at all. Physicochemical data have to be correlated with biological activity and the complexity of the living cell has to be reflected in the biophysical setups. This covers the use of full-length proteins instead of only functional domains, processed proteins instead of the straightforward bacterial expression, and the introduction of a membranous environment vs simple experiments in solution. 

By combining bacterial expression and chemical synthesis Ras constructs with the properties of the post-translationally modified protein can be generated. These hybrid proteins can insert into artifical and biological membranes, have been proven to be efficient tools for biochemical, biophysical and biological experiments and can be synthesized in large amounts. Principally the same method is applicable to many of the Ras-related GTP-binding proteins or the y-subunit of heterotrimeric G proteins. 

Based on the same principle, there are monomeric / -helical proteins that carry at their extremities a cluster of helical or nonrepetitive structures that could act as a capping element covering their exposed ends (Emsley et al., 1996 Lietzke et al, 1994 Petersen et al, 1997 Steinbacher et al, 1994). For example, the last 40 residues of pectate lyase C form a large loop that partially covers the surface of the /Hielix (Yoder et al, 1993). The fibrous (or otherwise elongated) domain of these natural /f-stranded proteins is not stable in isolation, as for example in the case of the P22 tailspike where bacterially expressed isolated /Hielix domain, at high concentrations, forms fibrous aggregates that bind Congo red (Schuler et al, 1999). 

The success of Chapman and co-workers in expression of flavocytochrome 2 in E. coli [23] is encouraging in its impUcations for future expression of flavoproteins in this host because, in their experience both the flavin and heme groups are incorporated into the recombinant protein. Moreover, the bacterial expression system produces the protein 500-1000 fold more efficiently than the yeast from which it was cloned. The enzyme produced in E. coli, however, lacks the first five amino acid residues at its amino terminus, a result which presumably reflects subtle differences in protein synthesis between the two organisms. 

A synthetic DNA cassette was designed that encoded the repeat sequence Lys-25 (Figure 2). This DNA monomer was synthesized, sequenced, and self-ligated to afford a mixture of DNA concatamers. A 3,000 base pair concatameric gene was iolated from this mixture, which encoded a polymer of Lys-25 with a molecular mass of approximately 90 kD. Bacterial expression of the concatameric gene afforded a protein of the expected molecular mass that accumulates to high levels in E. coli (Figure 3). 

With the development of bacterial expression systems for Mb described earlier and with the known diversity of metal coordination... 

Chang YJ, Song SH, Park SH, Kim SU. (2000) Amorpha-4,11-diene synthase of Artemisia annua cDNA Isolation and bacterial expression of a terpene synthase involved in artemisinin biosynthesis. Arch Biochem Biophys 3S3 178-184. 

Pharmacology Anakinra is a recombinant, nonglycosylated form of the human interleukin-1 receptor antagonist (IL-1 Ra). Anakinra differs from native human IL-IRa in that it has a single methionine residue at its amino terminus. It is produced by recombinant DNA technology using an Escherichia coli bacterial expression system. 

Steele CL, Crock J, Bohlmann J, Croteau R. (1998). Sesquiterpene synthases from grand fir Abies grandis). Comparison of constitutive and wound-induced activities, and cDNA isolation, characterization, and bacterial expression of delta-selinene synthase and gamma-humulene synthase./owrna/ of Biological Chemistry, 273,2078. 

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