Expression in E. coli

Expression in E. coli. For expression of a foreign DNA in E. coli it must be correctly transcribed and the transcript properly processed and translated. Such expression often requires the design of a suitable plasmid and DNA sequence, e.g., by insertion of an initiation codon before the coding region or by coupling of the gene to be expressed to the promoter for another operon. A sophisticated tailoring 

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Overexpression of apoaequorin (Inouye et al., 1989, 1991). To produce a large quantity of apoaequorin, an apoaequorin expression plasmid piP-HE containing the signal peptide coding sequence of the outer membrane protein A (ompA) of E. coli (Fig. 4.1.12) was constructed and expressed in E. coli. The expressed apoaequorin was secreted into the periplasmic space of bacterial cells and culture medium. The cleaving of ompA took place during secretion thus the... 

The gene of Aequorea GFP was cloned by Prasher et al. (1992), and expressed in E. coli and Caenorhabditis elegans by Chalfie et al. (1994) and in E. coli by Inouye and Tsuji (1994a). The X-ray structure of recombinant GFP was solved by Ormo et al. (1996) and Yang et al. (1996,1997). The protein is in the shape of a cylinder consisting of 11 strands of (3-sheets and an a-helix inside (which contains the chromophore), with short helical segments on the ends of the cylinder. Thus the chromophore is sealed and protected from the outside medium. 

A cDNA encoding apoobelin was obtained from O. longissima and sequenced (Illarionov et al., 1995). The deduced amino acid sequence of the apoobelin consists of 195 amino acid residues, with a calculated molecular mass of about 22.2 kDa, closely matching the apoproteins of other Ca2+-sensitive photoproteins such as aequorin from the jellyfish Aequorea (Inouye et al., 1985 Prasher et al., 1985) and clytin from the jellyfish Phialidium gregarium (Inouye and Tsuji, 1993). To obtain recombinant apoobelin, the cDNA encoding apoobelin was expressed in E. coli (Illarionov et al., 2000). The recombinant apoobelin produced was purified and converted into obelin by incubation with coelenterazine in the presence of molecular oxygen and 2-mercaptoethanol or dithioerythritol, as in the case of aequorin. 

The cDNA for this photoprotein has been cloned and expressed in E. coli, and the recombinant protein obtained was named mitrocomin (Fagan et al., 1993). Mitrocomin consists of 190 amino acid residues with a tyrosine residue at the C-terminus, and has three Ca2+-binding sites. 

The Rieske protein II (SoxF) from Sulfolobus acidocaldarius, which is part, not of a bci or b f complex, but of the SoxM oxidase complex 18), could be expressed in E. coli, both in a full-length form containing the membrane anchor and in truncated water-soluble forms 111). In contrast to the results reported for the Rieske protein from Rhodobacter sphaeroides, the Rieske cluster was more efficiently inserted into the truncated soluble forms of the protein. Incorporation of the cluster was increased threefold when the E. coli cells were subject to a heat shock (42°C for 30 min) before induction of the expression of the Rieske protein, indicating that chaperonins facilitate the correct folding of the soluble form of SoxF. The iron content of the purified soluble SoxF variant was calculated as 1.5 mol Fe/mol protein the cluster showed g values very close to those observed in the SoxM complex and a redox potential of E° = +375 mV 111). 

In the biosynthetic approach, protein expression in E. coli [27], yeast [28, 29], and plants [30, 31] have been employed. This approach requires the construction of genes encoding for these repetitive polypeptides. Different methods for gene construction have been pubhshed multimerization [32], recursive directional ligation [33], and recursive directional ligation via plasmid reconstruction [23, 34]. 

ALBRECHT M, KLEIN A, HUGUENEY p, SANDMANN G and KUNTZ M (1995) Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating -carotene desaturation , FEES Lett, 372, 199-202. 

LOTAN T and HIRSCHBERG J (1995) Cloning and expression in E. coli of the gene encoding 3-C-4-oxygenase, that converts (3-carotene to the ketocarotenoid canthaxanthin in Haematococcus pluvialis , FEBS Lett, 364, 125-8. 

The gene for the dehydratase was expressed in E. coli under lac promoter, and an expression plasmid pOxD 90F was constructed. The transformant was cultivated under optimal condition at 30° C when much of the enzyme was expressed in a soluble form with more than thousand and several hundred times than the wild-type strain per culture (up to more than 50% of the total soluble protein of the... 

Lowe, P. A. and Rhind, S. K., Solubilization, refolding, and purification of eukaryotic proteins expressed in E. coli, in Protein Purification — Micro to Macro, Burgess, R., Ed., Alan R. Liss, New York, 1988, 429. 

R)- and ( -selective HNLs. A number of recombinant HNLs have also been expressed in E. coli, Saccharomyces cerevisiae, and Pichia pastoris. Recently, protein engineering has been successfully applied to the development of a tailor-made HNL for large-scale production of specific cyanohydrins [69,70]. 

Disulfide bonds are hardly formed in cytosol of unmodified E. coli strains. Such enzymes can be expressed in E. coli Origami strain, in cells coexpressing helper proteins, such as PDI or DsbC, or they can be directed to the more oxidative periplasm. 

Scheich, C., Leitner, D., Sievert, V. et al. (2004) Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis. BMC Structural Biology, 4, 4. 

Fig. 10. Structure ofF. solani tpisi cutinase expressed in E. coli [121]. Locations of active site residues are indicated...

It is worth mentioning that metabolic engineering of E. coli recently provided recombinant strains which synthesized PHAMCL from gluconate. For this, beside phaC2Po or phaClPa> the thioesterase I from E. coli (TesA) [128] or the acyl-ACP thioesterase from Umbellularia californica [129], respectively, were expressed in E. coli. However, the amounts of PHAMCL accumulated in the cells were rather low, and this artificial pathway was not very efficient. 

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