Proteins Overexpressed in E. coli

Halobacterium salinurum membrane contains a family of four photoactive retinal proteins, called archaeal rhodopsins that are similar to our visual pigments in their 

It is noteworthy that the C CP-MAS NMR spectrum of the transmembrane a-helices from [l- C]Val-labeled ppR in egg PC bilayer is clearly visible, although the corresponding peaks from the loops are partly suppressed. Interestingly, the former peaks are appreciably suppressed in the presence of the transducer / HtrII (1-159) as revealed by the C CP-MAS NMR spectrum when compared with those of free / pR, leaving no spectral change in the loop regions. It is also notable that a 

The cognate transducer pYUvll is composed of a cytoplasmic domain divided into linker, methylation, and signaling regions, in addition to the transmembrane domain consisting of the two helices, TMl and The truncated transducer 

NMR can be assigned to the peak position of the an- helix (16.7-15.5 ppm) and its boundary with the ordinary aj -helix (15.5-15.0 ppm), with reference respectively to the chemical shifts of (Ala)n in hexafluoroisopropanol (HFIP) solution or solid 

The intense an-helical NMR peaks resonated at 16.7 and 16.3 ppm are clearly seen as a doublet peak for pHtrll (1-159) complexed with / pR, besides a broad envelope at 15.5 ppm, as recorded by the DD-MAS NMR (Fig. 35A) but 

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The availability of cDNAs has also greatly aided biochemical studies which can now utilize large quantities of highly purified protein overexpressed in E. coli and other heterologous systems, rather than relying on partially purified, low specific activity natural sources (Sandmann et al 1993 Misawa et al., 1994). While this allows individual enzyme activities to be studied in some detail it provides little information about integration and... 

Paulsen H, Riimler U and Rudiger W (1990) Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed in E. coli. Planta 181 204-211... 

Pognonec, P., Kato, H, Sumimoto, H, Kretzschmar, M, and Roeder, R. G (1991) A quick procedure for purification of functional recombinant proteins overexpressed in E.Coli Nucl Acid. Res 19, 6650... 

The nifU gene product (NifU) from A. vinelandii has been overexpressed in E. coli and the recombinant protein purified and characterized (60). NifU is a homodimer of 33-kDa subunits with 2 Fe atoms per subunit. Spectroscopic studies showed the presence of [Fe2S2l clusters with = 254 mV and only cysteinyl coordination, but with... 

Three other proteins with similar domain structure as that of FprA were reported in other bacteria (WasserfaUen et al. 1995 Gomes et al. 1997, 2000). The recombinant CthFprA and CthHrb, overexpressed in E. coli, were purified and characterized. Both FprA and Hrb were found to be present as dimers. Metal/cofactor analysis of the purified proteins revealed the presence of 2 mol each of iron and flavin (FMN) per mole dimer of Hrb and 4 mol of iron and 2 mol FMN per mole dimer of FprA. The EPR spectra of the purified proteins indicated that iron is present in a di-iron center in FprA and as a Fe(Cys)4 cluster in Hrb. 

Both BFD variants L476Q and 55E4 were overexpressed in E. coli BL21 (DE3) as His-tag fusion proteins and purified to electrophoretic homogeneity as detected by SDS-PAGE by metal chelate affinity chromatography using Ni-NTA (data not shown). 

The /V-formylmethionine of a nascent protein synthesized in bacteria is removed by the sequential activities of PDF and a methionine aminopeptidase to generate the mature protein. The gene encoding PDF was cloned and overexpressed in E. coli by Meinnel and coworkers (1993). The PDF enzyme has an unusual metal ion (Fe2+) as its catalyst. However, the ferrous ion in this enzyme is unstable and can be quickly and irreversibly oxidized to ferric ion, rapidly inactivating the enzyme. PDF-based assay development therefore depended on the ability of nickel ion to replace ferrous ion in vitro, increasing the stability of the enzyme and maintaining its enzymatic activity (Groche et al., 1998 Clements et al., 2001 Hackbarth et al., 2002). 

When recombinant proteins are overexpressed in E. coli, insoluble inclusion bodies are often formed. It has been observed that molecular chaperonin prevents insoluble body formation when it is coexpressed. Chaperonin activity can be examined by monitoring the decreasing rate of insoluble formation. The cobQ gene of KODl strain, which encodes cobyric acid synthase, leads to an insoluble inclusion complex when it is overexpressed in E. coli. The model experiment using CobQ as a target for CpkB is described below. 

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