Description of Research Group
We are interested in enzymes from extremophiles, especially those originating from cold environments. This includes understanding how these enzymes have evolved to function under particular conditions, and investigating their uses in biotechnological applications.
Our main project ‘Lig-Eng – Engineering DNA Ligases for Next-Generation-Sequencing’ aims to produce novel DNA ligase enzymes with superior properties that can be used in preparation of Next Generation Sequencing (NGS) DNA libraries. DNA ligases are enzymes that join pieces of DNA together- an essential step in preparing DNA samples for NGS by attaching small ‘adapter’ pieces of
DNA to it.
We use in silico function prediction and clustering based on sequence homology to select diverse wild-type enzyme candidates from publically-available and in-house databases, and express the proteins recombinantly in E. coli. Fluorescence-based enzyme assays (both end-point and real-time) are used to measure the efficiency of the recombinant enzymes in DNA ligation, and to determine substrate specificity with a particular focus on double-strand DNA breaks. These assays are also used to find optimal activity conditions such as temperature, pH, ionic strength and cofactor concentration, which are essential for integration of new enzymes into existing biotech workflows. Some ligases will be modified further by random mutagenesis and rational engineering, and key to this is the determination of high-resolution structures of the ligases in complex with their substrate DNA using X-ray crystallography.
Various biophysical methods are used to assess stability, substrate binding and the oligomeric state of the ligases including: differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF) gel filtralion (GF) and electrophoretic mobility shift assay (EMSA). Additional methods Biacore,
isothermal titration calorimetry (ITC) and Microscale Thermophoresis (MST) will be implemented in coming months
Additional research interests include in the group: the biological function and evolution of bacterial ATP-dependent DNA ligases (b-ADLs); determination of properties of ‘minimal’ b-ADLs from psychrophilic bacteria; studies of the ‘group 15’ carbohydrate esterases from marine bacteria; collaborations on various other industrially interesting enzymes from extremophiles, including alcohol dehydrogenases and proteases.
Technology, expertise and equipment
Technology/ methods:
- Recombinant protein expression and purification (especially cold-adap1ed proteins)
- Time-resolved enzyme assays (molecular beacon)
- DNA modification assays (denaturing urea PAGE with fluorescently labeled substrates)
- Size determination/ interaction (EMSA, GF)
- Thermal stability/ ligand binding (DSC, DSF)
- X-ray crysuillography
- 3rd generalion sequencing (Oxford nanopore)
Expertise:
- Low volume/ high value commercial enzymes
- DNA repair and DNA modification
- Structural biology
Equipment:
- Common equipment in Norstruct, (department of Chemistry, UiT) – no specialist equipment owned by this group.
