Difference between revisions of "Part:BBa K1582015"
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         <tr class="firstRow">
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             <td style="border: 1px solid windowtext; padding: 0px 7px;" valign="top" width="123">
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             <td style="border: 1px solid windowtext; padding: 0px 7px;" valign="top" width="277">
 
                 <p style="text-align:left">
 
                 <p style="text-align:left">
−
                     <span style="font-size:16px">0mM McAc</span>
+
                     <span style="font-size:19px">0mM McAc</span>
 
                 </p>
 
                 </p>
 
             </td>
 
             </td>
−
             <td style="border-width: 1px 1px 1px medium; border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="125">
+
             <td style="border-width: 1px 1px 1px medium; border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="277">
 
                 <p style="text-align:left">
 
                 <p style="text-align:left">
−
                     <span style="font-size:16px">Resuspension</span>
+
                     <span style="font-size:19px">Resuspension</span>
 
                 </p>
 
                 </p>
 
             </td>
 
             </td>
 
         </tr>
 
         </tr>
 
         <tr>
 
         <tr>
−
             <td style="border-width: medium 1px 1px; border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="123">
+
             <td style="border-width: medium 1px 1px; border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="277">
 
                 <p style="text-align:left">
 
                 <p style="text-align:left">
−
                     <span style="font-size:16px">10mM McAc</span>
+
                     <span style="font-size:19px">10mM McAc</span>
 
                 </p>
 
                 </p>
 
             </td>
 
             </td>
−
             <td style="border-width: medium 1px 1px medium; border-style: none solid solid non
+
             <td style="border-width: medium 1px 1px medium; border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">Wash the impurity away</span>
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td style="border-width: medium 1px 1px; border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">20mM McAc</span>
 +
                </p>
 +
            </td>
 +
            <td style="border-width: medium 1px 1px medium; border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">Wash the impurity away</span>
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td style="border-width: medium 1px 1px; border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">30mM McAc</span>
 +
                </p>
 +
            </td>
 +
            <td style="border-width: medium 1px 1px medium; border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">Wash the impurity away</span>
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td style="border-width: medium 1px 1px; border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">50mM McAc</span>
 +
                </p>
 +
            </td>
 +
            <td style="border-width: medium 1px 1px medium; border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">Wash the impurity away</span>
 +
                </p>
 +
            </td>
 +
        </tr>
 +
        <tr>
 +
            <td style="border-width: medium 1px 1px; border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; -moz-border-top-colors: none; -moz-border-right-colors: none; -moz-border-bottom-colors: none; -moz-border-left-colors: none; border-image: none; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">200mM McAc</span>
 +
                </p>
 +
            </td>
 +
            <td style="border-width: medium 1px 1px medium; border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; padding: 0px 7px;" valign="top" width="277">
 +
                <p style="text-align:left">
 +
                    <span style="font-size:19px">Elute</span>
 +
                </p>
 +
            </td>
 +
        </tr>
 +
   
 +
</table>
 +
[[File:Tianjin_wy.png]]
 +
'''Figure 19.''' BFP- sJanus-m: The target protein is about 40kDa. As is shown in the picture, the protein is purified through Ni-chelating affinity chromatography.

Revision as of 02:51, 25 September 2015

BFP+sJanus-m Fusion Protein


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 961
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 970


Usage

Admittedly the separation of proteins is difficult in biological experiments. More often than not, we use chromatography, but it is expensive. Extraction turns to be cheap, however, its low separation rate is really an obstacle. </br> Here comes the Janus, a kind of amphipathic protein, which is made into a fusion protein with the target and carry it to leave the bulk protein phase. We could use aqueous two-phase systems to make them separated from mixture, according to the property that Janus will direct to the phase of detergent in the system of detergent/polymer. BFP-inJanus fusion protein could be used to verify this purification system. </br>

Biology

mTagBFP is a monomeric protein with a narrow fluorescence emission spectrum with a maximum at 456 nm. It has a tyrosine-based chromophore, giving it substantially higher brightness, faster chromophore maturation and higher pH stability than blue fluorescent proteins with a histidine in the chromophore. </br> sJanus are small secreted fungal proteins which can be found in filamentous fungi, which play a role in a broad range of processes in the growth and development of filamentous fungi. They shows thread-like structure, can be expressed in prokaryotic cells like E.coli, and are involved in transferring themselves to detergent phase spontaneously. </br> We did some mutations to it, and we call it sJanus-m.</br>

Reference

[1]Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands. Hydrophobins: multipurpose proteins. Annu Rev Microbiol. 2001;55:625-46.


Protein Expression

We insert the plasmid into competent C+ to make the fusion protein secreted.</br> Express the fusion protein:</br>

5ml tube with C+

37℃ 15h

1L culture bottle

37℃ 4h

induced by IPTG

500μl  16℃ 12-16h

Collect the bacterial

Break the bacterial


Purify the fusion protein:

0mM McAc

Resuspension

10mM McAc

Wash the impurity away

20mM McAc

Wash the impurity away

30mM McAc

Wash the impurity away

50mM McAc

Wash the impurity away

200mM McAc

Elute

Tianjin wy.png Figure 19. BFP- sJanus-m: The target protein is about 40kDa. As is shown in the picture, the protein is purified through Ni-chelating affinity chromatography.