Asociación categórica de las variables
4.2 Discusión de resultados
Xu et al., 2008 developed the procedure used within the lab to produce NanB. This procedure used Luria Broth (LB) and isopropyl ß-D-1-thiogalactopyranoside (IPTG) to produce NanB protein from a PET23b (ampicillin-resistant) plasmid (for full procedure see Experimental). LB is the most commonly used media for E. coli as it is easy to make, rich in nutrients and contains the optimal osmolarity for growth in early log phase (Rosano and Ceccarelli, 2014). Despite these features it is not the best media for achieving high cellular density due to lack of carbohydrates and divalent cations (Rosano
Grubbs Catalyst
Loading (mol%) Time (hrs) Temperature (°C) Solvent Yield (%)
1st generation 0.05 3 RT DCM 51
1st generation 0.05 5 RT DCM 54
1st generation 0.05 3 40 Toluene 64
1st generation 0.05 5 40 Toluene 71
2nd generation 0.05 3 RT DCM 69
Grubbs 2nd generation catalyst Grubbs 1st generation catalyst
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and Ceccarelli, 2014). IPTG is one of the most commonly used chemical inducers of the lac operon within biochemical labs. This thio-galactoside is a mimic of allolactose and inhibits the lac repressor
lacI resulting in lac operon transcription (Politz et al., 2013). This chemical inducer is added during the mid-logarithmic phase of growth usually at an optical density (OD) at 600nm of 0.6 (Koopmans, 2009). Using this procedure 21 mg of NanB protein was expressed and purified from 4 liters of E. coli
LB bacterial culture.
Figure 36. SDS page of NanB purified through a nickel column. 1. Original expression method (IPTG induction) and 2. First optimization attempt (autoinduction).
To optimize this procedure it was reasoned that the autoinduction technique commonly used within the Taylor group would result in an improved yield of NanB. Autoinduction media was introduced in 2005 and is an optimized blend of glucose, lactose and glycerol. Glucose is metabolized first as the preferred carbon source, and then glycerol and lactose (an inducer of the lac operon) metabolism follows in mid to late-logarithmic phase. This negates the need for biomass monitoring, culture manipulation and manual addition of the inducer (avoiding human error) (Rosano and Ceccarelli, 2014). This enriched media can produce a yield of target protein several-fold higher than IPTG induction (Studier, 2005). Protein purity was evaluated after the first purification process (a nickel column) using an SDS-PAGE gel. The autoinduction procedure produced a cleaner batch of protein compared with expression in LB after the first purification process (Figure 36). The anion exchange purification was omitted from the procedure and the protein fractions were directly subjected to a final purification step (size exclusion chromatography). Using this optimized procedure 52 mg of protein was expressed and purified from 4 liters of E.Coli autoinduction media bacterial culture. This represents a 2.5 times increase in yield compared to the IPTG and LB broth procedure (21 mg). An
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increase of 1.2g in biomass was observed for the autoinduction method for each 1L of bacterial culture (this represents an increase of 27%).
In order to determine if the protein produced using this optimised method was useful for the kinetic analysis of inhibitors, it was compared against the NanB previously produced using the original method. Activities of neuraminidases/sialidases are tested using the 2’-(4-methylumbelliferyl)-α-D– N-acetylneuraminic acid (4-munana) assay. This assay is described by Kongkamnerd et al., 2011 and used to probe the activity of neuraminidases/sialidases. In the presence of a neuraminidase/sialidase, the substrate is cleaved into 30 and 4-methylumbelliferyl (4-Mu) (Kongkamnerd et al., 2011)
(
scheme 5). The fluorescence intensity of the fluorophore 4-Mu generated over time is measured giving the rate of the enzyme.Scheme 5. The substrate 4-munana cleaved into compounds 30 and 4-Mu.
The activity of NanB purified and expressed from each approach was tested using the 4-munana assay at the optimum conditions of 37°C and pH 5.0. The activity of NanB differed between the two methods of expression and purification. Autoinduction produced a less active batch of protein compared with the IPTG LB method (Table 4).
Table 4. Table of the enzymatic activity of NanB expressed and purified using the two approaches: IPTG (original method) and autoinduction (first attempt at optimization). Protein assayed using the 4-munana assay with 4-munana at a final concentration of 200µM and NanB at a final concentration of 60 ng/mL.
Protein Expression and
Purification method
Mean
activity
(AFU/sec)
Autoinduction 250±18
IPTG induction 1100±25
Based upon these results protein produced using the autoinduction method was used only for crystallization. For kinetic analysis, the IPTG method was chosen for NanB expression.
30 4-Mu
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An adaption to the purification method suggested by Professor Terry Smith involved purification of the protein without the use of cocktail inhibitors as this had shown to inhibit protein activity in the past. The purification method in the presence and absence of protease cocktail inhibitors was trialed. It was found that protein purified without protease cocktail inhibitors was more active (1440±120 AFU/second).
Excluding protease cocktail inhibitors from the method resulted in an increased activity of 340 AFU/sec. The next step was to assess carefully the
purification
steps. Protein activity was assessed at each stage of the purification procedure and observed to be the most active after the anion exchange purification step (1886±84 AFU/second) The purity of the protein after the anion exchange column by SDS page gel was observed to be similar to that after gel size exclusion chromatography (Figure 37). It was decided to avoid gel filtration and use protein directly after the anion exchange column.Figure 37. SDS Page of NanB after anion exchange purification (1) and size-exclusion chromatography (2).
Table 5. Table of the enzymatic activity of NanB after each purification step.
Purification Step
Mean activity (AFU/ second)
Nickel Column (step 1) 281±30
Desalt Column (step 2) 476±16
Anion Exchange Column (step 3) 1886±84
Gel Filtration Column (step 4) 1440±120
The stability of the enzyme was measured over time. Left at 4°C the enzymatic activity remains stable for one week and left at -80°C the enzymatic activity remains stable for a month.
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Having established an activity and stability profile of NanB, testing of Optactin against NanB was initiated.