Part:BBa_K3183203

mClover3 with SpyTag and 6-His for E. coli

mClover3 is a 26.9 kDa protein derived from GFP. mClover3 improves photostability by 60% (t_1/2 = 80s) to its predecessor, owing to 2 mutations relative to dClover2: A206K and S160C. mClover3 can be used in Förster Resonance Energy Transfer (FRET) experiments with mRuby3, providing an alternative to cyan/yellow partners. This has the advantage of reducing spectral separation, having lower phototoxicity, and lower autofluorescence¹.
This construct was used for Spy&Go purification as a part of our characterisation of SpyTag, BBa_K1159201. Expression of this part was achieved in the pET28A vector BBa_K2141000.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

Expression of this construct was used to verify the effectiveness of Spy&Go as a purification technique:

Documentation
We adapted [parts.igem.org/Part:BBa_K1159201 K1159201] as an affinity tag for our proteins. In our construct, we used the SpyTag as a secondary affinity tag. The purification method was developed by Anuar et al., 2019. It uses an immobilized protein, SpyDock which can bind SpyTag reversibly. SpyDock captures fusion tags and allows elution at high (2.5M) imidazole concentration. To assess the capabilities for purification, we used densitometric analysis to determine how pure our protein product is and how the purification relates to the widely-used 6xHis Ni-NTA purification method. We used a construct that consisted of mClover3 with SpyTag and 6xHis tag. Moreover, the purified protein was used to make a standard curve using fluorometric measurements. The standard curve enables the comparison of promoters that use mClover3 as a reporter protein.

Methods
The samples were expressed in the same conditions (BL21 (DE3) - RIPL E. coli expression strain, pET28a expression vector with T7 expression system, induced with IPTG (420mM final concentration) at OD600 = 0.6 and at 21C. The cultures were spun down and resuspended in lysis buffer (Spy & Go: 40mM Tris, 25mM Phosphoric acid, pH = 7; Ni-NTA: 30mM Tris, 300mM NaCl, 10mM imidazole for IMAC purification; both had PI (Roche), PMSF (1mM), benzonase (1U/mL), and hen egg white lysozyme (1mg/mL) added) and lysed using sonication. After centrifuging the lysate at 17000g for 10min, the clear lysate was incubated with 250uL packed resin volume in both cases and incubated for 1 hour before loading onto a gravity column. The columns were washed 4 times, and the protein was eluted with Ni-NTA elution buffer (200mM imidazole, 30mM Tris, 300mM NaCl) or Spy&Go elution buffer (2.5M imidazole, 40mM Tris, 25mM Phosphoric acid, pH = 7.00). 500μL fractions were collected and run on an SDS-PAGE (fig. 5 & 6).

Results

Figure 5: Spy&Go Affinity Purification using SpyTag-mClover3-6His

Figure 6: Ni-NTA Affinity Purification using SpyTag-mClover3-6His

Using BCA assay, we determined the total protein concentration of the samples. Then, the samples were loaded onto another SDS-PAGE gel in 0.5mg/ml concentration. The SDS-PAGE gel (run for 45 min, at 200V) was stained with Coomassie blue (InstantBlue™️) and then imaged using BioRad Chemidoc XRS and analysed using Image Lab (ver. 6.0.1, BioRad). In Image Lab, bands were detected and compared with the rest of the lane at background subtraction of disk size 2mm. Percentage purity is equivalent to Lane % (see fig. 7).

Figure 7: mClover3 Purification Comparison Between Ni-NTA and Spy&Go Using Densitometry

Figure 8: mClover3 standard curve - Fluorescence intensity vs Concentration (mg/ml). On the graph, we can observe that in MRS the FI is lower than that in PBS. This could be due to the high fluorescence of MRS that masks mClover3 fluorescence.Error bars represent 1 s.d., n=3

Spy&Go purification was able to purify the protein of interest with 60.1% (±4.4%, n=3) purity. This is lower than that of the Ni-NTA purification (74.5%, ±5.0%, n=3). Using this data and the results of BCA assay, we were able to determine that the total concentration of mClover3 from the Spy&Go purification was 1.0 mg/ml (±0.316mg/ml, n=3) from, while the Ni-NTA purification yielded1.5 mg/ml (±0.344mg/ml, n=3). From this, we were able to calculate the approximate yield. Spy&Go purification yielded a 8.2 mg/L (±2.525, n=3) of mClover3, whereas Ni-NTA purification yielded a 15.4mg/L of mClover3 (±3.438, n=3).

References

1) Bajar, Bryce T et al. “Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.” Scientific reports vol. 6 20889. 16 Feb. 2016, doi:10.1038/srep20889