Difference between revisions of "Part:BBa K2609007"

 
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<partinfo>BBa_K2609007 short</partinfo>
 
<partinfo>BBa_K2609007 short</partinfo>
  
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This part generates mcp-1 (a mouse monocyte chemokine) when expressed inside a T7 expression strain like Bl21 (DE3). The protein is fused to a 6xHis tag and a TEV protease cleavage site on the N-term.
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This part generates Mcp-1 (a mouse monocyte chemokine) when expressed inside a T7 expression strain like BL21 (DE3). The protein is fused to a 6xHis tag and a TEV protease cleavage site at the N-term. Mcp-1 is a member of CC chemokine family that uses the CCR2 receptor for chemo-attracting monocytes to the site of release. This coding sequence lacks the heavily glycosylated C-terminus present in the wild-type protein and has been shown to have a increased chemotactic potency<sup>[1][2]</sup>. The protein can be expressed recombinantly in a prokaryotic system because of its lack of glycosylation and post-translational modifications.
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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<html>
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<h2>Biology</h2>
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Mcp-1 belongs to the CC family of chemokines and is recognised by the CCR2 receptors found on murine monocytes. Due to its inherent homology to human CCL2, it acts as a effective chemokine for human moncytes as well. <b>The N-term region of the protein has been implicated in receptor binding and any fusion to this terminal might result in decreased activity. </b>
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In both mice and humans, the chemokine is produced by a wide variety of cell types including endothelial,fibroblasts, epithelial, smooth muscle, mesangial, astrocytic, monocytic, and microglial cells. Apart from monocytes, CCL2 has also been shown to chemoattract NK cells and T cells with comparatively lesser efficacy.<sup>[2]</sup>
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<h2>Usage</h2>
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For their Phage Assisted Immune Recruitment (<a href="http://2018.igem.org/Team:IISc-Bangalore/PAIR">PAIR</a>) system, the 2018 IISc-Bangalore iGEM team used the mcp-1 CDS fused with both N-term and C-term secretion tags (PelB and HlyA respectively) for recombination into a phage genome. This engineered phage will recruit phagocytic monocytes to the site of infection and prevent release of bacterial toxins directly into the body.
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</html>
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==Characterization==
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<html>
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<figure style="float: right; width: 35%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em;">
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<img src="https://static.igem.org/mediawiki/parts/0/05/T--IISc-Bangalore--Registry_mcp-1Exp.png" width=100% style="border: 1px solid black;"></img>
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<figcaption><a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a> expressed in BL21 (DE3) at 25°C for 16 hrs. 1mL of the culture was resuspended in 100uL PBS and mixed with 100uL of 2xSDS Sample Buffer followed by boiling. 20uL of the final mix was loaded onto a SDS-PAGE gel.</figcaption>
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</figure>
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<h3>Expression with <a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a></h3>
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<p>The protein was expressed under T7 promoter in E. coli BL21 (DE3) with a 6xHis tag connected to the N-term through a TEV protease cleavage site. Small scale induction was done at the temperatures 37°C, 25 °C and 16°C and IPTG concentrations ranging from 100uM to 1mM. The protein was found in inclusion bodies at all temperature with negligible yield at lower temperatures (See 'Solubilization'). The ideal conditions for expression after multiple trials expression were found to be the following:</p>
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<ul>
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<li>Temperature: 25°C
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<li>IPTG Concentration: 250uM
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<li>Time of induction: 16 hrs (Not optimized)
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<li>Chloramphenicol concentration: 35ug/ml (Not optimized)
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<li>Medium: LB (Not optimized)
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</ul>
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The protein with the 6xHis and TEV protease tags should be 15.6kDa in size. Bands were seen between 17kDa and 22kDa after induction.
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<h3 style="clear:right;">Solubilization with <a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a></h3>
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At the favorable conditions for high yield, the protein expressed from <a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a> in a flask culture was insoluble. We postulated the formation of inclusion bodies, which are not uncommon when an eukaryotic protein is expressed in <i>E. coli</i>. Formation of inclusion bodies was checked by resuspension of the cell pellet in lysis buffers containing different detergents.
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<figure style=" width: 80%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: auto; padding: 0.5em;">
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<img src="https://static.igem.org/mediawiki/parts/6/6e/T--IISc-Bangalore--mcp1diffDet.png" width=100% style="border: 1px solid black;"></img>
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<figcaption>Cell pellet from BL21 (DE3) after expression (25°C, 250uM IPTG, 16hrs) in <a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a> was solubilized in different different detergents. Lane 1 and 2: Cell pellet; Lane 3: Ladder; Lane 4 and 5: Lysis buffer with no detergent; Lane 6 and 7: Lysis buffer with 1% Triton X-100; Lane 8 and 9: Lysis buffer with 2% SDS.</figcaption>
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</figure>
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<br>
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Small amounts of soluble protein was obtained only with 2% SDS lysis buffer. Hence, <b>the protein forms inclusion bodies when expressed in BL21 (DE3)</b>. The absence of soluble protein was also noted at both 16°C (24 hrs, 250uM IPTG) and 37°C (3 hrs, 250uM IPTG).
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The inclusion from the cell pellet were solubilized in lysis buffer containing 8M urea for subsequent purification.
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<figure style="float:right; width: 60%; text-align: center; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em;">
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<img src="https://static.igem.org/mediawiki/parts/f/fc/T--IISc-Bangalore--mcp1Purification.png" width=100% style="border: 1px solid black;"></img>
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<figcaption>Cell pellet from BL21 (DE3) after expression (25°C, 250uM IPTG, 16hrs) in <a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a> was solubilized in different different detergents. Lane 1 and 2: Cell pellet; Lane 3: Ladder; Lane 4 and 5: Lysis buffer with no detergent; Lane 6 and 7: Lysis buffer with 1% Triton X-100; Lane 8 and 9: Lysis buffer with 2% SDS.</figcaption>
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</figure>
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<h3>Purification with <a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609007</a></h3>
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After solubilization in 8M urea, the protein was directly used for a Ni-NTA purification and the purified protein (in 8M Urea) was dialysed against buffers containing decreasing concentration of urea for refolding. Both the solubilization and refolding protocols can be found on the IISc-Bangalore Team's <a href="http://2018.igem.org/Team:IISc-Bangalore/Protocols">protocols page</a>.
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</html>
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 19:03, 10 October 2018


mcp-1 under T7 expression system

This part generates Mcp-1 (a mouse monocyte chemokine) when expressed inside a T7 expression strain like BL21 (DE3). The protein is fused to a 6xHis tag and a TEV protease cleavage site at the N-term. Mcp-1 is a member of CC chemokine family that uses the CCR2 receptor for chemo-attracting monocytes to the site of release. This coding sequence lacks the heavily glycosylated C-terminus present in the wild-type protein and has been shown to have a increased chemotactic potency[1][2]. The protein can be expressed recombinantly in a prokaryotic system because of its lack of glycosylation and post-translational modifications.


Usage and Biology

Biology

Mcp-1 belongs to the CC family of chemokines and is recognised by the CCR2 receptors found on murine monocytes. Due to its inherent homology to human CCL2, it acts as a effective chemokine for human moncytes as well. The N-term region of the protein has been implicated in receptor binding and any fusion to this terminal might result in decreased activity. In both mice and humans, the chemokine is produced by a wide variety of cell types including endothelial,fibroblasts, epithelial, smooth muscle, mesangial, astrocytic, monocytic, and microglial cells. Apart from monocytes, CCL2 has also been shown to chemoattract NK cells and T cells with comparatively lesser efficacy.[2]

Usage

For their Phage Assisted Immune Recruitment (PAIR) system, the 2018 IISc-Bangalore iGEM team used the mcp-1 CDS fused with both N-term and C-term secretion tags (PelB and HlyA respectively) for recombination into a phage genome. This engineered phage will recruit phagocytic monocytes to the site of infection and prevent release of bacterial toxins directly into the body.

Characterization

BBa_K2609007 expressed in BL21 (DE3) at 25°C for 16 hrs. 1mL of the culture was resuspended in 100uL PBS and mixed with 100uL of 2xSDS Sample Buffer followed by boiling. 20uL of the final mix was loaded onto a SDS-PAGE gel.

Expression with BBa_K2609007

The protein was expressed under T7 promoter in E. coli BL21 (DE3) with a 6xHis tag connected to the N-term through a TEV protease cleavage site. Small scale induction was done at the temperatures 37°C, 25 °C and 16°C and IPTG concentrations ranging from 100uM to 1mM. The protein was found in inclusion bodies at all temperature with negligible yield at lower temperatures (See 'Solubilization'). The ideal conditions for expression after multiple trials expression were found to be the following:

  • Temperature: 25°C
  • IPTG Concentration: 250uM
  • Time of induction: 16 hrs (Not optimized)
  • Chloramphenicol concentration: 35ug/ml (Not optimized)
  • Medium: LB (Not optimized)
The protein with the 6xHis and TEV protease tags should be 15.6kDa in size. Bands were seen between 17kDa and 22kDa after induction.

Solubilization with BBa_K2609007

At the favorable conditions for high yield, the protein expressed from BBa_K2609007 in a flask culture was insoluble. We postulated the formation of inclusion bodies, which are not uncommon when an eukaryotic protein is expressed in E. coli. Formation of inclusion bodies was checked by resuspension of the cell pellet in lysis buffers containing different detergents.
Cell pellet from BL21 (DE3) after expression (25°C, 250uM IPTG, 16hrs) in BBa_K2609007 was solubilized in different different detergents. Lane 1 and 2: Cell pellet; Lane 3: Ladder; Lane 4 and 5: Lysis buffer with no detergent; Lane 6 and 7: Lysis buffer with 1% Triton X-100; Lane 8 and 9: Lysis buffer with 2% SDS.

Small amounts of soluble protein was obtained only with 2% SDS lysis buffer. Hence, the protein forms inclusion bodies when expressed in BL21 (DE3). The absence of soluble protein was also noted at both 16°C (24 hrs, 250uM IPTG) and 37°C (3 hrs, 250uM IPTG). The inclusion from the cell pellet were solubilized in lysis buffer containing 8M urea for subsequent purification.
Cell pellet from BL21 (DE3) after expression (25°C, 250uM IPTG, 16hrs) in BBa_K2609007 was solubilized in different different detergents. Lane 1 and 2: Cell pellet; Lane 3: Ladder; Lane 4 and 5: Lysis buffer with no detergent; Lane 6 and 7: Lysis buffer with 1% Triton X-100; Lane 8 and 9: Lysis buffer with 2% SDS.

Purification with BBa_K2609007

After solubilization in 8M urea, the protein was directly used for a Ni-NTA purification and the purified protein (in 8M Urea) was dialysed against buffers containing decreasing concentration of urea for refolding. Both the solubilization and refolding protocols can be found on the IISc-Bangalore Team's protocols page. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 464
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]