Difference between revisions of "Part:BBa K2926048"
Line 27: Line 27:
 
<figure  >
 
<figure  >
 
  <img src="https://2019.igem.org/wiki/images/7/7e/T--Bielefeld-CeBiTec--mCherry_Structure.png" style="width:50%" >
 
  <img src="https://2019.igem.org/wiki/images/7/7e/T--Bielefeld-CeBiTec--mCherry_Structure.png" style="width:50%" >
−
  <figcaption> <b> Crystal structure of mCherry (Shu and Remington 2006).</b><br>
+
  <figcaption> <b> Fig.&nbsp;&nbsp;1: Crystal structure of mCherry (Shu and Remington 2006).</b><br>
 
  </figcaption>
 
  </figcaption>
 
  </figure>
 
  </figure>
Line 50: Line 50:
  
 
<h1>Protein purification</h1>
 
<h1>Protein purification</h1>
−
The part mCherryHis was expressed, purified and characterized together with the parent part mCherry (BBa_J06504).<br>
+
The part mCherryHis was expressed, purified and characterized together with the parent part mCherry (<a href="https://parts.igem.org/Part:BBa_J06504"> BBa_J06504 </a> ).<br>
 +
To further characterize purified mCherry we compared two different purification protocols. Purification
 +
via his-tag was compared to the IMPACT-purification protocol from NEB. For this purpose we cloned mCherry
 +
( <a href="https://parts.igem.org/Part:BBa_J06504"> BBa_J06504 </a> ) into the purification and expression vector pTXB1 from NEB and at the same time added six
 +
histidines to the C-terminus of mCherry in pSB1C3 ( <a href="https://parts.igem.org/Part:BBa_K2926048"> BBa_K2926048 </a> ). Both expression vectors were transformed
 +
in <i>E. coli </i> ER2566. After induction with IPTG both cultures showed the characteristic red colour of mCherry
 +
expressing bacteria (Fig.&nbsp;&nbsp;2 and Fig.&nbsp;&nbsp;3).
 +
</div>
 +
<div class="middle">
 +
<figure >
 +
<img class="figure image" src="https://2019.igem.org/wiki/images/3/30/T--Bielefeld-CeBiTec--mCherry_Cultures.png" style="width:75%">
 +
<figcaption> <b> Fig. 2: Expression cultures of mCherry in pTXB1 (left) and mCherryHis in pSB1C3 (right).</b><br>
 +
                                        Expression cultures of mCherry in pTXB1 and mCherryHis in pSB1C3 in <i>E. coli</i> ER2566 were cultivated to an OD of
 +
                                        around 0.6 at 37&nbsp;&nbsp;°C in LB with 100&nbsp;&nbsp;mg Ampicillin per L. Protein expression was induced by addition of IPTG to a final
 +
                                        concentration of 0.4&nbsp;&nbsp;mM. After additional 30&nbsp;&nbsp;min at 37&nbsp;&nbsp;°C cultures were transferred to 17&nbsp;&nbsp;°C and protein expression
 +
                                        was performed over night.
 +
</figcaption>
 +
</figure> <br>
 +
</div>
 +
                                    <div class="">
 +
                                    The expression culture of mCherry in pTXB1 showed a brighter red colour which indicated a higher expression level.
 +
                                    </div>
 +
<div class="middle">
 +
<figure class="figure large">
 +
<img class="figure image" src="https://2019.igem.org/wiki/images/d/dc/T--Bielefeld-CeBiTec--mCherry_Harvested.png" style="width:75%">
 +
<figcaption> <b> Fig. 3: Harvested expression cultures of mCherry in pTXB1 (left) and mCherryHis in pSB1C3 (right).</b><br>
 +
Expression cultures of mCherry and mCherryHis in <i>E. coli</i> ER2566 were harvested by centrifugation at
 +
4&nbsp;&nbsp;°C for 20&nbsp;&nbsp;min and 4&nbsp;&nbsp;000&nbsp;&nbsp;rpm.
 +
</figcaption>
 +
</figure> <br>
 +
</div>
 +
<div class="">
 +
 
 +
<h1>Purification</h1>
 +
 
 +
After cultivation and cell lysis via Ribolyzer the protein was purified using the His-purification kit from Macherey Nagel
 +
and the IMPACT-purification kit from NEB (Fig.&nbsp;&nbsp;4).
 +
</div>
 +
<div class="middle">
 +
<figure >
 +
<img class="figure image" src="https://2019.igem.org/wiki/images/a/ab/T--Bielefeld-CeBiTec--mCherry_purification-columns.png" style="width:75%">
 +
<figcaption> <b> Fig. 4: Purification columns for IMPACT- (left) and Ni-TED-purification (right).</b><br>
 +
Harvested cells were lysed using Zirconia metal beads (1&nbsp;&nbsp;mm) in a Ribolyzer at 8&nbsp;&nbsp;000&nbsp;&nbsp;rpm for 15&nbsp;&nbsp;s. The lysate
 +
was cleared by centrifugation at 4&nbsp;&nbsp;°C for 1&nbsp;&nbsp;h and 4&nbsp;&nbsp;500&nbsp;&nbsp;rpm. Cleared lysate was loaded onto a chitin column
 +
(IMPACT-purification) or a Ni-TED column (purification via his tag) and washed with wash buffer. Finally the protein
 +
was eluted, washed in PBS and concentrated.
 +
</figcaption>
 +
</figure> <br>
 +
</div>
 +
<div class="">
 +
A performed Bradford assay showed that expression and purification using the IMPACT-Kit resulted in a higher yield since
 +
we were able to purify 985&nbsp;&nbsp;µg mCherry from a cell mass of 2.13&nbsp;&nbsp;g compared to 39.4&nbsp;&nbsp;µg mCherryHis from a cell mass of 1.92&nbsp;&nbsp;g.
 +
Both purification methods were analyzed on a SDS-PAGE (Fig.&nbsp;&nbsp;5).
 +
</div>
 +
<div class="middle">
 +
<figure>
 +
<img src="https://2019.igem.org/wiki/images/c/cf/T--Bielefeld-CeBiTec--mCherry_SDS-PAGE_MALDI.png" style="width:75%">
 +
<figcaption> <b> Fig. 5: SDS-PAGE of the protein purification.</b><br> <i>E. coli</i> lysate of the expression culture,
 +
flow-through- and wash-fraction as well as the purified protein were denatured by heating the samples to 98&nbsp;&nbsp;°C
 +
for 10&nbsp;&nbsp;min in SDS-PAGE loading buffer containing DTT and loaded on an polyacrylamide-gel (12&nbsp;&nbsp;%). The proteins
 +
were separated through electrophoresis (25&nbsp;&nbsp;mA). Suggested mCherry bands in the lane with purified proteins were
 +
marked in dark red.
 +
</figcaption>
 +
</figure> <br>
 +
</div>
 +
<div class="">
 +
The SDS-PAGE shows an intense band at the estimated height of around 27&nbsp;&nbsp;kDa in every lane. This indicates that mCherry
 +
as well as mCherryHis have successfully been expressed. The bands in the wash- and flow-through-fraction show, that not
 +
all of the protein efficiently binds to the purification columns. <br>
 +
                                      In the last lane you can see that we were able to purify mCherry as well as mCherryHis. While the IMPACT-purification
 +
resulted in a higher yield, the purity of mCherryHis was higher as the protein lane in Fig. 5 indicated. <br>
 +
Following the SDS-PAGE we analyzed the purified protein via MALDI-ToF. For this purpose we excised the marked bands (Fig.&nbsp;&nbsp;5)
 +
from the SDS-PAGE and started a tryptic digestion of the washed gel fragment. Analysis via MALDI-ToF confirmed that we were
 +
able to purify mCherry (Fig.&nbsp;&nbsp;6).
 +
</div>
 +
<div class="">
 +
<div class="middle">
 +
<figure >
 +
<img class="figure image" src="https://2019.igem.org/wiki/images/3/38/T--Bielefeld-CeBiTec--mCherry_MALDI.png" style="width:75%">
 +
<figcaption> <b> Fig.&nbsp;&nbsp;6: Mass spectrum of mCherry (1) and mCherryHis (2) after tryptic digestion.</b><br>
 +
Excised bands from the SDS-PAGE of mCherry and mCherryHis were washed, digested over night with trypsine and
 +
co-cristallyzed with a HCCA-matrix on a MALDI target. Mass spectrum was recorded in a MALDI-ToF MS from Bruker
 +
Daltronics and data was evaluated using the software BioTools.
 +
</figcaption>
 +
</figure> <br>
 +
</div>
 +
<div class="">
 +
The generated mass spectra and mass lists were evaluated using the software BioTools. To compare the experimentally
 +
determined data to the theoretical protein sequence we performed an in silico trypsine-digestion of the expected protein
 +
sequence and compared the generated mass spectrum and mass list to the measured ones. We were able to match both proteins
 +
to the theoretical spectrum. Additionally we were able to detect the his-tag from mCherryHis in the mass list.
 +
</div>
 +
 
 +
<h1 id="a4">Characterization</h1>
 +
 
 +
To gain some more knowledge about mCherry we analyzed different properties of the protein. First of all we measured its
 +
emission- and excitation spectra (Fig.&nbsp;&nbsp;7).
 +
</div>
 +
<div class="middle">
 +
<figure >
 +
<img class="figure image" src="https://2019.igem.org/wiki/images/0/06/T--Bielefeld-CeBiTec--mCherry_Spectra.png" style="width:75%">
 +
<figcaption> <b> Fig.&nbsp;&nbsp;7: Emission- and excitation spectra of mCherry.</b><br> Emission- (dashed lines) and excitation-spectra
 +
(solid lines) of mCherry purified via IMPACT-Kit (dark purple) and His-tag (pink) were measured (λ<sub>Ex</sub>=570&nbsp;&nbsp;nm,
 +
λ<sub>Em</sub>=610&nbsp;&nbsp;nm) using the TECAN infinite M200 and normalized to their maximum.
 +
</figcaption>
 +
</figure> <br>
 +
</div>
 +
<div class="">
 +
The resulting spectra show, that adding a his-tag to mCherry does not alter the emission- or excitation spectrum of mCherry.
 +
The excitation maximum of mCherry lies at 587&nbsp;&nbsp;nm, the emission maximum at 608&nbsp;&nbsp;nm. <br><br>
 +
Next we compared the fluorescence intensity of the two different mCherry-variants normalized to Texas Red (Fig.&nbsp;&nbsp;8).
 +
 
 +
</div>
 +
<div class="middle">
 +
<figure >
 +
<img class="figure image" src="https://2019.igem.org/wiki/images/a/a2/T--Bielefeld-CeBiTec--mCherry_Intensities.png" style="width:75%">
 +
<figcaption> <b> Fig. 8: Fluorescence intensity of the dilution series of the two mCherry variants.</b><br> Fluorescence intensity of
 +
a dilution series of mCherry purified via IMPACT-Kit (dark purple) and mCherryHis (pink) was measured
 +
(λ<sub>Ex</sub>=570&nbsp;&nbsp;nm, λ<sub>Em</sub>=610&nbsp;&nbsp;nm) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5&nbsp;&nbsp;µM Texas Red
 +
at the same wavelength.
 +
</figcaption>
 +
</figure> <br>
 +
</div>
 +
<div class="">
 +
The fluorescence intensity of mCherryHis seemed to be higher than the intensity of mCherry purified via IMPACT kit.
 +
This might be due to the different purification protocols. Cleavage of mCherry from the chitin column during the
 +
IMPACT-purification is mediated through incubation of the column for 20-24&nbsp;&nbsp;h in DTT at room temperature. Those
 +
purification conditions might have a negative impact on the protein. Compared to Texas Red, the fluorescence intensity of
 +
1&nbsp;&nbsp;µmol mCherryHis equals the fluorescence intensity of 1.92&nbsp;&nbsp;µmol of the fluorescent dye. In contrast, the fluorescence
 +
intensity of 1&nbsp;&nbsp;µmol mCherry purified via IMPACT protocol equals the fluorescence intensity of 565&nbsp;&nbsp;nmol Texas Red. <br> <br>
 +
 +
 
 +
</div>
 
</html>
 
</html>

Revision as of 21:04, 17 October 2019


mCherry with hexahistidine tag for purification

This part codes for the red fluorescent protein mCherry with a C-terminal hexahistidine tag for simple purification via metal ions.

Usage and Biology

The red fluorescent protein mCherry was fused to a hexahistidine tag to enable easy purification. Since the first successful cloning of the green fluorescent protein GFP of Aequorea victoria in 1992 (Prasher et al. 1992) fluorescent proteins became a widely used tool in many fields of research. In contrast to antibodies labeled with fluorophores that have to cross the cellular membrane which severely disturbes the cellular integrity, flourescing proteins enable live cell imaging and the investigation of native states of the cell.
Because of the wide range of applications for fluorescing proteins there was a great interest in finding and engineering improved variants and a wider colour spectrum. In the last few years red fluorescing proteins became more and more important. Common native red fluorescing proteins are often dimeric or tetrameric what makes their usage in experimental setups difficult. Directed mutation of dsRFP from the corallimorpharia Discosoma sp. Led to the first monomeric red fluorescing protein mRFP1 (Shaner et al. 2004). Unfortunately this mutations resulted in a lower quantum yield and decreased photostability (Shaner et al. 2004). During further protein engineering attempts, scientists were able to create the red fluorescent protein mCherry. mCherry is a 26.7  kDa protein that shows a very short maturation time of about 15  minutes and a low acid sensitivity. Its excitation maximum lies at 587  nm and it has its emission maxiumum at 610  nm (www.fpbase.org). In 2006 the crystal structure of mCherry was published (Shu and Remington 2006).
Fig.  1: Crystal structure of mCherry (Shu and Remington 2006).
mCherry consists of 13 beta-sheets which form a beta-barrel and three alpha helices. The chromophore is made of methionine, tyrosine and glycine who posttranslationally form an imidazolinone (Shu et al. 2006).

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]

Sequence was validated by Sanger sequencing

Protein purification

The part mCherryHis was expressed, purified and characterized together with the parent part mCherry ( BBa_J06504 ).
To further characterize purified mCherry we compared two different purification protocols. Purification via his-tag was compared to the IMPACT-purification protocol from NEB. For this purpose we cloned mCherry ( BBa_J06504 ) into the purification and expression vector pTXB1 from NEB and at the same time added six histidines to the C-terminus of mCherry in pSB1C3 ( BBa_K2926048 ). Both expression vectors were transformed in E. coli ER2566. After induction with IPTG both cultures showed the characteristic red colour of mCherry expressing bacteria (Fig.  2 and Fig.  3).
Fig. 2: Expression cultures of mCherry in pTXB1 (left) and mCherryHis in pSB1C3 (right).
Expression cultures of mCherry in pTXB1 and mCherryHis in pSB1C3 in E. coli ER2566 were cultivated to an OD of around 0.6 at 37  °C in LB with 100  mg Ampicillin per L. Protein expression was induced by addition of IPTG to a final concentration of 0.4  mM. After additional 30  min at 37  °C cultures were transferred to 17  °C and protein expression was performed over night.

The expression culture of mCherry in pTXB1 showed a brighter red colour which indicated a higher expression level.
Fig. 3: Harvested expression cultures of mCherry in pTXB1 (left) and mCherryHis in pSB1C3 (right).
Expression cultures of mCherry and mCherryHis in E. coli ER2566 were harvested by centrifugation at 4  °C for 20  min and 4  000  rpm.

Purification

After cultivation and cell lysis via Ribolyzer the protein was purified using the His-purification kit from Macherey Nagel and the IMPACT-purification kit from NEB (Fig.  4).
Fig. 4: Purification columns for IMPACT- (left) and Ni-TED-purification (right).
Harvested cells were lysed using Zirconia metal beads (1  mm) in a Ribolyzer at 8  000  rpm for 15  s. The lysate was cleared by centrifugation at 4  °C for 1  h and 4  500  rpm. Cleared lysate was loaded onto a chitin column (IMPACT-purification) or a Ni-TED column (purification via his tag) and washed with wash buffer. Finally the protein was eluted, washed in PBS and concentrated.

A performed Bradford assay showed that expression and purification using the IMPACT-Kit resulted in a higher yield since we were able to purify 985  µg mCherry from a cell mass of 2.13  g compared to 39.4  µg mCherryHis from a cell mass of 1.92  g. Both purification methods were analyzed on a SDS-PAGE (Fig.  5).
Fig. 5: SDS-PAGE of the protein purification.
E. coli lysate of the expression culture, flow-through- and wash-fraction as well as the purified protein were denatured by heating the samples to 98  °C for 10  min in SDS-PAGE loading buffer containing DTT and loaded on an polyacrylamide-gel (12  %). The proteins were separated through electrophoresis (25  mA). Suggested mCherry bands in the lane with purified proteins were marked in dark red.

The SDS-PAGE shows an intense band at the estimated height of around 27  kDa in every lane. This indicates that mCherry as well as mCherryHis have successfully been expressed. The bands in the wash- and flow-through-fraction show, that not all of the protein efficiently binds to the purification columns.
In the last lane you can see that we were able to purify mCherry as well as mCherryHis. While the IMPACT-purification resulted in a higher yield, the purity of mCherryHis was higher as the protein lane in Fig. 5 indicated.
Following the SDS-PAGE we analyzed the purified protein via MALDI-ToF. For this purpose we excised the marked bands (Fig.  5) from the SDS-PAGE and started a tryptic digestion of the washed gel fragment. Analysis via MALDI-ToF confirmed that we were able to purify mCherry (Fig.  6).
Fig.  6: Mass spectrum of mCherry (1) and mCherryHis (2) after tryptic digestion.
Excised bands from the SDS-PAGE of mCherry and mCherryHis were washed, digested over night with trypsine and co-cristallyzed with a HCCA-matrix on a MALDI target. Mass spectrum was recorded in a MALDI-ToF MS from Bruker Daltronics and data was evaluated using the software BioTools.

The generated mass spectra and mass lists were evaluated using the software BioTools. To compare the experimentally determined data to the theoretical protein sequence we performed an in silico trypsine-digestion of the expected protein sequence and compared the generated mass spectrum and mass list to the measured ones. We were able to match both proteins to the theoretical spectrum. Additionally we were able to detect the his-tag from mCherryHis in the mass list.

Characterization

To gain some more knowledge about mCherry we analyzed different properties of the protein. First of all we measured its emission- and excitation spectra (Fig.  7).
Fig.  7: Emission- and excitation spectra of mCherry.
Emission- (dashed lines) and excitation-spectra (solid lines) of mCherry purified via IMPACT-Kit (dark purple) and His-tag (pink) were measured (λEx=570  nm, λEm=610  nm) using the TECAN infinite M200 and normalized to their maximum.

The resulting spectra show, that adding a his-tag to mCherry does not alter the emission- or excitation spectrum of mCherry. The excitation maximum of mCherry lies at 587  nm, the emission maximum at 608  nm.

Next we compared the fluorescence intensity of the two different mCherry-variants normalized to Texas Red (Fig.  8).
Fig. 8: Fluorescence intensity of the dilution series of the two mCherry variants.
Fluorescence intensity of a dilution series of mCherry purified via IMPACT-Kit (dark purple) and mCherryHis (pink) was measured (λEx=570  nm, λEm=610  nm) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5  µM Texas Red at the same wavelength.

The fluorescence intensity of mCherryHis seemed to be higher than the intensity of mCherry purified via IMPACT kit. This might be due to the different purification protocols. Cleavage of mCherry from the chitin column during the IMPACT-purification is mediated through incubation of the column for 20-24  h in DTT at room temperature. Those purification conditions might have a negative impact on the protein. Compared to Texas Red, the fluorescence intensity of 1  µmol mCherryHis equals the fluorescence intensity of 1.92  µmol of the fluorescent dye. In contrast, the fluorescence intensity of 1  µmol mCherry purified via IMPACT protocol equals the fluorescence intensity of 565  nmol Texas Red.