Showing papers by "Neil R. Gilkes published in 1988"
TL;DR: Two enzymes prepared from Escherichia coli expressing recombinant DNA of the cellulolytic bacterium Cellulomonas fimi cleaved both enzymes in vivo in a highly specific manner, implying a critical role for the binding domain of CenA in the hydrolysis of crystalline substrate.
337 citations
TL;DR: The yeast MEL1 gene for secreted alpha-galactosidase was used to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae to show the yeast version to have an identical K(m) and pH optimum but to be more thermostable.
Abstract: We used the yeast MEL1 gene for secreted α-galactosidase to construct cartridges for the regulated expression of foreign proteins from Saccharomyces cerevisiae. The gene for a Cellulomonas fimi β-1,4-exoglucanase was inserted into one cartridge to create a fusion of the α-galactosidase signal peptide to the exoglucanase. Yeast transformed with plasmids containing this construction produced active extracellular exoglucanase when grown under conditions appropriate to MEL1 promoter function. The cells also produced active intracellular enzyme. The secreted exoglucanase was N-glycosylated and was produced continuously during culture growth. It hydrolyzed xylan, carboxymethyl cellulose, 4-methylumbelliferyl-β-d-cellobiose, and p-nitrophenyl-β-d-cellobiose. A comparison of the recombinant S. cerevisiae enzyme with the native C. fimi enzyme showed the yeast version to have an identical Km and pH optimum but to be more thermostable.
56 citations
TL;DR: Transcription of the cenA gene of Cellulomonas fimi, encoding endoglucanase A (EngA), from the lac promoter of pUC18 rather than from the tet promoter ofpBR322 increased the level of expression of the gene in Escherichia coli some 800-fold, resulting in the temperature-dependent leakage of periplasmic proteins, including EngA and β-lactamase, into the culture medium.
Abstract: Transcription of the cenA gene of Cellulomonas fimi, encoding endoglucanase A (EngA), from the lac promoter of pUC18 rather than from the tet promoter of pBR322 increased the level of expression of the gene in Escherichia coli some 800-fold. Most of the EngA activity was exported to the periplasm, even though the EngA leader peptide in the construct was 9 amino acids longer than usual. The increased level of expression of cenA resulted in the temperature-dependent leakage of periplasmic proteins, including EngA and β-lactamase, into the culture medium. In contrast to C. fimi, which processes pro-EngA at a single site, E. coli processed it at two distinct sites 3 amino acids apart. Increasing the level of expression of cex, encoding an exoglucanase (Exg) of C. fimi, to a comparable extent also led to the temperature-dependent leakage of Exg into the culture medium.
39 citations
Patent•
08 Jul 1988TL;DR: In this article, a fusion protein is prepared containing a polypeptide such as an enzyme and an amino acid sequence having a substrate binding region of a polysaccharidase such as cellulase.
Abstract: A fusion protein is prepared containing a polypeptide such as an enzyme and an amino acid sequence having a substrate binding region of a polysaccharidase such as cellulase that has essentially no polysaccharidase activity. By contacting the fusion protein with an affinity matrix containing a substrate such as cellulose for the cellulase substrate binding region, the substrate binding region binds to the affinity matrix to immobilize the polypeptide. The polypeptide can be purified by separating the fusion protein or polypeptide from the affinity matrix.
13 citations