4.3.2.1 Analysis of protein samples using 1D (1H) NMR
1D spectra of TgMIC3-EGF23 and TgMIC3-CE2 were collected from unlabelled shake-flask cultures of TgMIC3. TgMIC3-EGF234 originated from both SMD1168 and GS115 expression strains and were resistant up to 1.5 mg/ml Zeocin™ whilst TgMIC3- CE2 originated from both SMD1168 and X33 expression strains and were resistant up to 0.5 mg/ml Zeocin™. The protein samples were prepared using the method described in section 4.3.1.1.
The 1D (1H) NMR spectra of TgMIC3-CE2 shows a strong methyl peak at -0.1 ppm (Fig 4.15). However, both the amide region and the region from 5 - 6 ppm have no obvious peaks. This could indicate that the TgMIC2-CE2 construct could be partially folded.
The 1D (1H) NMR spectra of EGF234 shows no obvious peaks below 0 ppm (Fig 4.16), however this could be due to the lack of ring-shifted methyl groups in the structure. The presence of peaks in the amide region greater than 9 ppm indicates a folded domain. In addition, the peaks around 5 to 6 ppm arise due to Hα resonances in a β-sheet domain which provides further evidence of a folded domain.
Both TgMIC3-EGF3 and TgMIC3-CE2 showed promising spectral characteristics that could signify that both constructs could potentially be candidates for structural determination via NMR.
169 Figure 4.15: 1D (1H) NMR Spectrum of TgMIC3-CE2. A is an expanded view of the methyl region;
B is an expanded view of the amide region. There is a peak at -0.1ppm which is highly indicative of a folded domain. There are no obvious peaks in the amide region, which could indicate a partially folded domain. Spectra was collected at 308 K
170 Figure 4.16: 1D (1H) NMR Spectrum of TgMIC3-EGF234. A is an expanded view of the methyl
region; B is an expanded view of the amide region. The absence of peaks below 0 ppm could be due to the lack of ring shifted methyl groups whilst the presence of peaks in the amide region indicate that there is a folded region. Spectra was collected at 308 K
171 4.3.2.2 Analysis of protein samples using 2D (1H/15N) NMR
Labelled culture of TgMIC3 clones were produced using shake flask culture and according to the method described in section 4.13.1. TgMIC3-EGF234 originated from the SMD1168 expression strain and was resistant up to 1.5 mg/ml Zeocin™ whilst TgMIC3-CE2 originated from the SMD1168 expression strain and was resistant up to 0.5 mg/ml Zeocin™. TgMIC3-EGF234 had a purified protein yield of 0.12 mg/L whilst TgMIC3-CE2 had a purified protein yield of 0.02 mg/L.
(1H/15N) HSQC spectra of TgMIC3-CE2 exhibited promising spectral properties (Fig 4.17). However, some peak overlap was seen in the centre of the spectrum. This was speculated to be due to the presence of an unfolded domain within the protein. Similarly, the (1H/15N) HSQC spectra of TgMIC3-EGF234 also exhibited some promising spectral properties (Fig. 4.18). However, peaks were not as well dispersed as those in the TgMIC3-CE2 spectrum. 13C labelling via shake flask culture would require the use of an estimated 60-300 ml of 13C-methanol to produce a sufficiently concentrated sample for use in triple resonance experiments.
172 Figure 4.17: (1H/15N) HSQC Spectrum of TgMIC3-CE2. The peaks in the spectrum are generally well
dispersed, which indicates that the protein was relatively well folded. Spectrum was collected at 308 K with a field strength of 600 MHz.
173 Figure 4.18: (1H/15N) HSQC Spectrum of TgMIC3-EGF234. The peaks in the spectrum seem to be
gathered between 8.0 to 8.5 ppm, which could indicate the presence of a flexible region within the protein. Spectrum was collected at 308 K with a field strength of 600 MHz.
174 4.3.2.3 Crystallization of protein samples
Crystallization trials of both TgMIC3-CE2 and TgMIC3-EGF234 were conducted. In addition, protein samples for full length constructs of TgMIC3 and TgMIC4 were also produced and used for crystallization trials. Crystallization trials and optimizations for TgMIC3-EGF234, full-length TgMIC3 and full-length TgMIC4 were conducted using a protein sample produced from a single production run. The protein yield from a single production run of TgMIC3-CE2 was too low and necessitated the use of multiple batches of protein for crystallization trials.
All protein samples were confirmed to be homogenous and monodisperse via size exclusion chromatography - multi-angle light scattering (SEC-MALS) (Fig 4.19). All proteins were purified via gel filtration, concentrated to 40mg/ml and buffer exchanged into 10 mM Tris-HCl pH 7.0 immediately prior to initial crystallization trials. Crystallization trials were conducted at 4 °C and 20 °C.
4.3.2.3.1 Domain constructs of TgMIC3
The average yield for a single 4L fermentation run for TgMIC3-EGF234 was 3.5 mg. Crystallization trials for TgMIC3-EGF234 were conducted at 5, 10, 15 and 20 mg/ml protein concentrations but did not yield any hits. It was postulated that incomplete removal of sugar moieties might have precluded crystallization. Another batch of TgMIC3-EGF234 produced in the presence of tunicamycin to inhibit glycosylation was used for further crystallization trials but did not yield any hits.
The average yield for a single 4L fermentation run for TgMIC3-CE2 was 0.32 mg. Crystallization trials for TgMIC3-CE2 were conducted at 5, 10, 20, 40 and 60 mg/ml. Crystals were obtained in one condition at 4 °C using a protein concentration of 40 mg/ml. The initial crystallization condition was 0.1M NaCl, 0.1M HEPES pH7.5, 1.6M (NH4)2SO4.
175 Figure 4.19: SEC-MALS of TgMIC3 Full Length. Peak limits were defined as 1.255 - 1.433 ml.
Results indicated that the sample was less than 1% polydisperse, an average molar mass moment of 25000 Da and an average root mean squared radius moment of 5.9 nm.
176
A
B
C
Figure 4.20: Crystals of TgMIC4. A is a crystal of full length TgMIC4 which diffracted to 9Å.
Conditions for crystal growth were 35% (v/v) 2-methyl-2,4-pentanediol, Na/K phosphate pH 6.2 with a protein concentration of 10 mg/ml (Wizard I - 43). B is a crystal of full length TgMIC4 which diffracted to 8Å. Conditions for crystal growth were 35% (v/v) 2-methyl-2,4-pentanediol, Tris pH 7.0, 0.2M NaCl with a protein concentration of 10 mg/ml (Wizard I – 24). C is a crystal of full length TgMIC4 which diffracted to 8Å. Conditions for crystal growth were 0.1 M Potassium chloride, 0.01 M Magnesium chloride hexahydrate, 0.05 M Tris-hydrochloride pH 8.5, 30% v/v Polyethylene glycol 400 with a protein concentration of 10 mg/ml (Natrix – 47). Crystals were obtained in 96 well plates.
177
4.3.2.3.2 Full length constructs of TgMIC3 and TgMIC4
The average yield for a single 4L fermentation run for full length TgMIC3 was 1.2 mg. Crystallization trials for full length TgMIC3 were conducted at 5, 10, 15 and 20 mg/ml protein concentrations but did not yield any hits. It was postulated that similarly to TgMIC3-EGF234, incomplete removal of sugar moieties had precluded crystallization. However, SEC-MALS data indicated that the sample was less than 1% polydisperse. A batch of full length TgMIC3 was produced in the presence of tunicamycin was used for further crystallization trials but did not yield any hits.
The average yield for a single 4L fermentation run for full length TgMIC4 was 2.8 mg. Crystallization trials for full length TgMIC4 were conducted at 5, 10, 15 and 20 mg/ml. Full details of all conditions tested can be found in the appendix Crystals were obtained in three conditions at 20 °C using a protein concentration of 10 mg/ml. Hits were obtained from formulation 43 from the Wizard I random sparse matrix crystallization screen (Emerald BioSystems), formulation 24 from the Wizard II random sparse matrix crystallization screen (Emerald BioSystems) and formulation 57 from the Natrix™ screen (Hampton Research) (Fig. 4.20). Optimization of crystals was attempted using vapour drop diffusion methods. Crystals were reproduced in similar conditions but did not exhibit any visible improvement in size. Crystals obtained diffracted to 8 Å resolution.
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4.4 Conclusion
Protein production using P. pastoris as an expression system was successful in producing homogenous, monodisperse protein samples for specific constructs such as Full length TgMIC3 and TgMIC4. However, for hyperglycosylated samples such as TgMIC3-EGF234, complete and uniform removal for sugar moieties proved difficult to achieve and further study is needed to develop a repeatable and effective protocol for uniform deglycosylation. Full length TgMIC4 was successfully crystallized and diffracted to a resolution of 8Å. TgMIC3-CE2 produced crystals but protein yields were insufficient for further investigation. 15N labeled protein samples were produced for TgMIC3-EGF234 and TgMIC3-CE2 and successfully used to gather 2D (1D/15N) HSQC spectra. However, the expected yield and suboptimal spectral qualities did not justify moving on to 13C labeling and subsequent triple resonance experiments.
179