Difference between revisions of "Part:BBa K3185000"
Line 33: Line 33:
 
</ul>
 
</ul>
 
<h3><font size="4.5">SDS-PAGE</font></h3>
 
<h3><font size="4.5">SDS-PAGE</font></h3>
−
[[File:SPYtag TmEncapsulin.png|800px|thumb|left|alt text]]
+
[[File:SPYtag TmEncapsulin.png|300px|thumb|right|alt text]]
 
<br>
 
<br>
 
<br>
 
<br>
 
==Result==
 
==Result==
−
[[File:peak191015.png|300px|thumb|left|Fig. 1 TmEncapsulin polymer appears as the peak.
+
[[File:peak191015.png|300px|thumb|right|Fig. 1 TmEncapsulin polymer appears as the peak.
 
<br>
 
<br>
 
TmEncapsulin expressed E. coli lysate and purified protein solution was loaded on 10%-60% sucrose linear gradient / 20 mM Tris 7.5, 50 mM NaCl, then centrifuged in 100,000g for 18 hours at 4℃ with SW41(Beckman).  The solution was fractionated on a 96-well plate with BioComp.  At the same time, 260nm absorption was measured. ]]
 
TmEncapsulin expressed E. coli lysate and purified protein solution was loaded on 10%-60% sucrose linear gradient / 20 mM Tris 7.5, 50 mM NaCl, then centrifuged in 100,000g for 18 hours at 4℃ with SW41(Beckman).  The solution was fractionated on a 96-well plate with BioComp.  At the same time, 260nm absorption was measured. ]]
 
<br>
 
<br>
 
<br>
 
<br>
−
[[File:191001 SPYt-SPYC 2.png|300px|thumb|left|Fig. 2 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
+
[[File:191001 SPYt-SPYC 2.png|300px|thumb|right|Fig. 2 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
 
<br>
 
<br>
 
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature.  Then 6µL of 2x SDS sample buffer was added.  10µL of each sample was loaded.  SDS-PAGE for 30min in 200V.  The gel was CBB stained.]]
 
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature.  Then 6µL of 2x SDS sample buffer was added.  10µL of each sample was loaded.  SDS-PAGE for 30min in 200V.  The gel was CBB stained.]]
 
<br>
 
<br>
 
<br>
 
<br>
−
[[File:SpyC-T timeD.png|300px|thumb|left|Fig. 3  Quantification of conjugated band
+
[[File:SpyC-T timeD.png|300px|thumb|right|Fig. 3  Quantification of conjugated band
 
<br>
 
<br>
 
Conjugated bands’ intensity was quantified with ImageJ.  Orange dots show averages value of three experiments.  Blacklines show standard deviations.  The time point 60min was deleted because it includes negative value.
 
Conjugated bands’ intensity was quantified with ImageJ.  Orange dots show averages value of three experiments.  Blacklines show standard deviations.  The time point 60min was deleted because it includes negative value.

Revision as of 23:38, 20 October 2019


SPYtag inserted Tm Encapsulin

Usage and Biology

TmEncapsulin is a protein found from Thermotoga maritima. A paper says that it consists of 60 monomers and forms capsule, Virus-like particle(VLP)[1]. iGEM also treats it as a useful part (BBa_K192000).

We used TmEncapsulin as a biological polymer. We inserted SpyTag in TmEncapsulin. This enables TmEncapsulin to display different types of proteins on the surface of the protein capsule. (See Fig.2) (SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201)[2]. SpyTag forms an isopeptide bond with SpyCatcher when they are mixed[3]. In previous research about TmEncap, it is showed peptides inserted after 138th amino acid in TmEncap can be exposed on the protein capsule as a loop[4]. Furthermore, “Author et.al” showed when SpyTag is inserted at the same position, SpyCatcher/SpyTag also forms a bond between SpyCatcher and SpyTag inserted TmEncap (SpyTmEnc)[5].
File:T--Kyoto--SpyTmEnc.png
Also, this has three tag and cleavage sites. First is 6x-His tag placed in the C-terminus of TmEncapsulin for protein purification by using Ni-NTA beads. However, in a paper, Ni-NTA beads cannot bind to 6x-His tag added in C-terminus because it doesn’t display enough to the surface of the protein capsule[4]. To solve this problem, we inserted second tag. Second is HAtag inserted between TmEncapsulin and 6x-His tag in expectation of C-terminus to display on the surface of the capsule. Third is 6x-His-tag and linker inserted between #43 and #44 amino acids of native encapsulin for improving heat-resistance of TmEncapsulin. To design third one, we refered BBa_K2686002 of iGEM EPFL 2018 and the same paper. (BBa_K2686002)



We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of SpyCatcher inserted TmEncapusulin by using SDS-PAGE.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 77
    Illegal BglII site found at 597
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 426
    Illegal SapI.rc site found at 457

Purification


Expression

  • Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
  • Protein was expressed in 0.1mM IPTG for 2hours.

SDS-PAGE

alt text



Result

Fig. 1 TmEncapsulin polymer appears as the peak.
TmEncapsulin expressed E. coli lysate and purified protein solution was loaded on 10%-60% sucrose linear gradient / 20 mM Tris 7.5, 50 mM NaCl, then centrifuged in 100,000g for 18 hours at 4℃ with SW41(Beckman). The solution was fractionated on a 96-well plate with BioComp. At the same time, 260nm absorption was measured.



Fig. 2 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained.



Fig. 3 Quantification of conjugated band
Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it includes negative value.

References

1 Putri, R.M., Allende-Ballestero, C., Luque, D., Klem, R., Rousou, K.A., Liu, A., Traulsen, C.H.H., Rurup, W.F., Koay, M.S.T., Castón, J.R., et al. (2017).
Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake.
ACS Nano 11, 12796–12804.

2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016).
Programmable polyproteams built using twin peptide superglues.
Proc. Natl. Acad. Sci. U. S. A. 113, 1202–1207.

3 Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., and Howarth, M. (2012).
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
Proc. Natl. Acad. Sci. U. S. A. 109.

4 Moon, H., Lee, J., Min, J., and Kang, S. (2014).
Developing genetically engineered encapsulin protein cage nanoparticles as a targeted delivery nanoplatform.
Biomacromolecules 15, 3794–3801.

5 Bae, Y., Kim, G.J., Kim, H., Park, S.G., Jung, H.S., and Kang, S. (2018).
Engineering Tunable Dual Functional Protein Cage Nanoparticles Using Bacterial Superglue.
Biomacromolecules 19, 2896–2904.