Team:Manchester/Notebook
Lab Book
So much... So much happened here. And so much is about to.- Dr. Walter Bishop
FRINGE
Polyphosphate Kinase Fusion Protein Kinetics
Basic aim: assess impact localisation tag has on kinetics
Introduction/background
Our project initially aimed to screen a selection of bacterial polyphosphate kinases to select one with the greatest forward rate of reaction (polyphosphate formation). Due to time constrain, we have decided to focus on a single polyphosphate kinase that we identified in the Brenda database. It’s from Corynebacterium glutamicum, a bacteria used for industrial production of amino acids. The bacteria was discovered to accumulate high concentrations of polyphosphate (600mM, 37% cell volume), which was largely attributed to a class II Polyphosphate kinase. This PPK (encoded by NCgl2620) has a Kcat value of 74 for ATP ➝ Poly P under the experimental conditions used. It is also fully soluble, unlike the E. coli PPK which was encapsulated within a Pdu microcompartment by Warren et al. earlier this year. We hypothesise that solubility may enhance efficiency of encapsulation.
At this stage we will only be trialing one localisation tag (PduD1-20) - we will characterise the other tags using mCherry and fluorescence microscopy as an indicator for incorporation into BMC.
We were planning on ordering these sequences as Gblocks and simultaneously cloning them into both the submission vector and the expression vector pNIC-bsa28 in frame with a T7 promoter. Then we can over-express these fusion proteins in BL21 (DE3) cells and purify them using a nickel affinity resin column.
After expression and purification, the enzymes constructs will be characterised via Michaelis-Menten kinetics using the Promega ADP-Glo TM Kinase Assay, a well-plate based assay that is suitable for ‘virtually any kinase’. Technical support from Promega confirmed that the kit should work on a polyphosphate kinase. The readout for the test is based on luciferase. We managed to secure a sponsorship of 400 assays kit from Promega.
Kinetics data generated from these assays will allow us to assess any impact the localisation tag or mCherry tag has on enzyme activity. Results from this will aid how we build the construct to target the enzyme into the BMC at a later stage.
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