High activity of inclusion bodies formed in <i>Escherichia coli</i> overproducing <i>Clostridium thermocellum</i> endoglucanase D

Tokatlidis, Kostas; Dhurjati, Prasad; Millet, J; Béguin, Pierre; Aubert, Jean-Paul

doi:10.1016/0014-5793(91)80478-l

Cited by 103 publications

(60 citation statements)

References 16 publications

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“…In both constructs, the third codon preceding the first codon of the duplicated segment was fused to the XhoI site created by mutagenizing the stop codon of ceMC. Both proteins could be isolated as intact forms from inclusion bodies, which provided protection against E. coli proteases (19,20). The molecular mass of CelC-DSCipA was 49 kDa (Fig.…”

Section: Resultsmentioning

confidence: 99%

Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA

et al. 1994

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The binding specificity of the duplicated segments borne by Clostridium thermocellum endoglucanase CelD and by the cellulosome-integrating protein CipA was investigated. The fusion protein CelC-DSCelD, in which the duplicated segment of CelD was fused to the COOH terminus of endoglucanase CelC, bound with an affinity of 4.7 x 10(7) M-1 to the fusion protein MalE-RDCipA, in which the seventh receptor domain of CipA was grafted onto the COOH terminus of the Escherichia coli maltose-binding protein MalE. The affinity of CelC-DSCelD for the homologous chimeric protein MalE-RDORF3p, carrying the receptor of the surface protein ORF3p, was 6.9 x 10(6) M-1. The fusion protein CelC-DSCipA, in which the duplicated segment of CipA was grafted onto the COOH terminus of CelC, did not bind detectably to MalE-RDCipA or MalE-RDORF3p. However, Western blotting (immunoblotting) experiments indicated that the duplicated segment of CipA was able to bind to a set of C. thermocellum proteins which are different from those recognized by the duplicated segment of CelD. These results argue against the hypothesis that ORF3p interacts with the duplicated segment of CipA. More probably, ORF3p binds to individual cellulases and hemicellulases harboring duplicated segments.

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Section: Resultsmentioning

confidence: 99%

Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA

et al. 1994

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“…Evidently, this is not the case for the in vivo aggregation of interleukin 10; for this protein, self-association must involve an almost completely native intermediate. Similarly, inclusion bodies formed by cellulase consist of essentially native protein and exhibit full enzymatic activity (Tokatlidis et al, 1991). It appears that there are at least 2 classes of inclusion bodies: the first class results from the association of nativelike intermediates as is the case for interleukin 10 and cellulase; the second class of inclusion bodies, which is represented by TEM P-lactamase, involves the association of a compact state having extensive secondary structure, but not necessarily nativelike tertiary interactions.…”

Section: Discussionmentioning

confidence: 99%

Folding and aggregation of TEM β‐lactamase: Analogies with the formation of inclusion bodies in Escherichia coli

et al. 1994

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The enzyme TEM @-lactamase has been used as a model for understanding the pathway leading to formation of inclusion bodies in Escherichia coli. The equilibrium denaturation of TEM p-lactamase revealed that an intermediate that has lost enzymatic activity, native protein fluorescence, and UV absorption, but retains 60% of the native circular dichroism signal, becomes populated at intermediate (1 .O-1.4 M) concentrations of guanidium chloride (GdmCl). This species exhibits a large increase in bis-1-anilino-%naphthalene sulfonic acid fluorescence, indicating the presence of exposed hydrophobic surfaces. When TEM @-lactamase was unfolded in different initial concentrations of GdmCl and refolded to the same final conditions by dialysis a distinct minimum in the yield of active protein was observed for initial concentrations of GdmCl in the 1.0-1.5 M range. It was shown that the lower reactivation yield was solely due to the formation of noncovalently linked aggregates. We propose that the aggregation of TEM @-lactamase involves the association of a compact state having partially exposed hydrophobic surfaces. This hypothesis is consistent with our recent findings that TEM @-lactamase inclusion bodies contains extensive secondary structure (Przybycien TM, Dunn JP, Valax P, Georgiou G, 1994, Protein Eng 7: 131-136).Finally, we have also shown that protein aggregation was enhanced at higher temperatures and in the presence of 5 mM dithiothreitol and was inhibited by the addition of sucrose. These conditions exert a similar effect on the formation of inclusion bodies in vivo. Keywords: 0-lactamase; inclusion bodies; molten globule; protein foldingIn recent years it has become apparent that the tendency of proteins to aggregate is of considerable significance for the physiology of the cell as well as for numerous applications in biotechnology (DeYoung et al., 1993). Protein aggregates are formed in vivo as a result of mutations that affect the folding pathway, expression of heterologous polypeptides, or exposure of the cell to certain environmental stresses. Expression of heterologous genes in Escherichia coli and other gram-negative bacteria is often accompanied by the formation of micronsize aggregates or inclusion bodies (Mitraki & King, 1989

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“…CelF, the CelS equivalent in C. cellulolyticum, also displays significant activity in reducing the viscosity of a CMC solution (271). Some of the cloned endoglucanase genes are celA (26), celB (105), celC (298), celD (143,326), celE (113), celF (245), celG (188), celH (361), celI (101,123,373), celJ (encoding component S2 of the complex) (1,8), celM (162), celN (373), celQ (9), celT (172), and celX (113).…”

Section: Genes and Enzymesmentioning

confidence: 99%

Cellulase, Clostridia, and Ethanol

Demain

Newcomb

2005

Microbiol Mol Biol Rev

804

579

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SUMMARY Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.

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High activity of inclusion bodies formed in Escherichia coli overproducing Clostridium thermocellum endoglucanase D

Cited by 103 publications

References 16 publications

Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA

Recognition specificity of the duplicated segments present in Clostridium thermocellum endoglucanase CelD and in the cellulosome-integrating protein CipA

Folding and aggregation of TEM β‐lactamase: Analogies with the formation of inclusion bodies in Escherichia coli

Cellulase, Clostridia, and Ethanol

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