Jean-Michel Masson

Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.

Inteins of Thermococcus fumicolans DNA Polymerase Are Endonucleases with Distinct Enzymatic Behaviors

The DNA polymerase gene of Thermococcus fumicolans harbors two intein genes. Both inteins have been produced in Escherichia coli and purified either as naturally spliced products from the expression of the complete DNA polymerase gene or directly from the cloned inteins genes. Both recombinant inteins exhibit endonuclease activity, with an optimal temperature of 70 °C. The Tfu pol-1 intein, which belongs to the Psp KOD pol-1 allelic family, recognizes and cleaves a minimal sequence of 16 base pairs (bp) on supercoiled DNA with either Mn2+ or Mg2+ as cofactor. It cleaves linear DNA only with Mn2+ and requires a 19-bp minimal recognition sequence. The Tfu pol-2 intein, which belongs to the Tli pol-2 allelic family, is a highly active homing endonuclease using Mg2+ as cofactor. Its minimal recognition and cleavage site is 21 bp long either on linear or circular DNA substrates. Its endonuclease activity is strongly inhibited by the 3′ digestion product, which remains bound to the enzyme after the cleavage reaction. According to current nomenclature, these endonucleases were named PI-TfuI and PI-TfuII. These two inteins thus exhibit different requirements for metal cofactor and substrate topology as well as different mechanism of action.

34 Applications of Extremophiles: The Industrial Screening of Extremophiles for Valuable Biomolecules

Publisher Summary This chapter explores in a non-exhaustive manner, the various screening approaches for the discovery of molecules with industrial interest, with a special focus on the screening of extremozymes. The search for new enzymes requires the development of suitable methods and assays for screening the catalytic activities in environmental samples, in pure cultures, or for identifying an enzyme during a molecular cloning step. The screening process should closely mimic the targeted conditions to rapidly result in the selection of an enzyme that fulfils the specifications defined by the industry. With the recent development of microbiology, chemistry, and molecular biology technologies, the field of enzyme screening and optimization is now experiencing major R&D initiatives and concrete results. Screening for enzymes active under extreme conditions, require suitable screening assays to detect activities under these conditions with a satisfactory signal-to-noise ratio. Expression cloning is a technology of choice for enzyme screening. Molecular screening of genomic libraries is achieved using well-known hybridization approaches.

Development of a reporter system for the yeast Schwanniomyces occidentalis: influence of DNA composition and codon usage

In this paper we report on searching for suitable reporters to monitor gene expression and protein secretion in the amylolytic yeast Schwanniomyces occidentalis. Several potential reporter and marker genes, formerly shown to be functional in other yeasts, were cloned downstream from the homologous invertase gene (INV) promoter and their activity was followed in conditions of repression and derepression of the INV promoter. However, neither β‐glucuronidase nor β‐lactamase nor phleomycin resistance‐conferring gene, all originating from E. coli, were expressed in S. occidentalis cells to such a level to allow for monitoring of their activity. All the reporter genes tested have a higher percentage of GC (47–62%) in their DNA compared to the DNA composition of S. occidentalis genes that are more AT‐rich (36% GC). The codon usage of all the reporter genes also varies from that of 16 so far sequenced S. occidentalis genes. This suggests that an appropriate composition of DNA and a codon usage similar to S. occidentalis genes might be very important parameters for an efficient expression of a heterologous gene in Schwanniomyces occidentalis. Indeed, two genes originating from Staphylococcus aureus, with an AT‐content in their DNA similar to that of S. occidentalis, were functionally expressed in S. occidentalis cells. Both a phleomycin resistance‐conferring gene and a chloramphenicol acetyltransferase‐encoding gene thus represent suitable reporters of gene expression and protein secretion in S. occidentalis. Additionally, we show in this work that the transcription‐regulating region and the signal peptide sequence of the S. occidentalis invertase gene were efficient to direct gene expression and subsequent protein secretion in Saccharomyces cerevisiae. Copyright

Production of a full length Tat protein in E. coli and its purification.

A full length tat gene was constructed by a combination of a polymerase chain reaction (PCR) for the first exon and chemical synthesis for the second exon. This gene was expressed in E. Coli under the control of the strongly regulated araB promoter, either directly or fused to a secretion signal encoding sequence. We the defined a rapid, three‐step procedure for the purification of the Tat protein

ARS sequences in homologous and heterologous ADE2 loci are capable of promoting autonomous replication of plasmids in Schwanniomyces occidentalis.

Abstract We have analyzed ARS elements linked to homologous and heterologous ADE2 loci functioning in Schwanniomyces occidentalis. We have identified a region of the ADE2 locus of S. occidentalis which promotes autonomous replication of plasmids in S. occidentalis cells. This region is within 385 bp preceding the ATG codon of the S. occidentalis ADE2 gene. It contains sequences similar to ARS core consensus sequences, ARS boxes, and a potential transcription activator binding site characterized in Saccharomyces cerevisiae. The ADE2 gene of S. cerevisiae was found to complement the ade2 mutation in S. occidentalis cells and the 5′ UTR region of this gene is capable of supporting autonomous replication of plasmids in S. occidentalis. Furthermore, we confirmed that the origin of replication of the 2 μm plasmid and the ARS1 sequence of S. cerevisiae are also functional in S. occidentalis cells. Plasmids carrying either ARS, the SwARSA element of S. occidentalis, the ARS linked to the ADE2 gene of S. cerevisiae, and the ARS1 sequence or the 2 μm ori, were found to be maintained in S. occidentalis cells as episomal monomers or oligomers. However, their stability was low as already reported for the ARS in S. occidentalis.

Cell-Free Protein-Based Enzyme Discovery and Protein–Ligand Interaction Study

Cell-free expression-based screening is sometimes more suitable than cell-based assays for enzyme discovery. The advantage of cell-free systems for expression of toxic, poorly expressed, or insoluble proteins has already been well documented. Cell-free methods can advantageously replace cell-based ones when screening has to be performed on cell lysates prepared from harvested cells, for instance, when dealing with protein-ligand interactions particularly when the latter is hydrophobic. From our experience, cell-free extracts efficient in both transcription and translation can be prepared from potentially any microorganism. Here we present a general method for preparation of cell-free extracts from prokaryotic and eukaryotic cells, selection of the best systems, and optimized conditions for specific protein expression. The method allows to select proteins for their ability to bind a selected target, to identify the inhibitors of such binding, or to identify novel enzymatic activities.