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Part:BBa_K883151

Designed by: Jeroen Bosman   Group: iGEM12_Wageningen_UR   (2012-09-19)

CCMV coat protein gene 25AA N-terminal replaced with HIStag under IPTG inducable promotor

This part produces a coat protein of the Cowpea Clorotic Mottle Virus (CCMV), with the first 25 amino acids replaced with a Histidine tag (6x).

Usage and Biology

CCMV is an abriviation of the Cowpea Chlorotic Mottle Virus, one of the most studied viruses in producing Virus Like Particles (VLPs). This part produces a coat protein of the Cowpea Clorotic Mottle Virus (CCMV), with the first 25 amino acids replaced by a Histidine tag (6x). When expressed in Escherichia coli, these coat proteins can be harvested and be assembled in vitro using the Wageningen_UR iGEM 2012 protocols.

Protocols for production and purification:

  • [http://2012.igem.org/Team:Wageningen_UR/Protocol/StartupCCMV| CCMV production protocol part 1]
  • [http://2012.igem.org/Team:Wageningen_UR/Protocol/DialysisCCMV| CCMV production protocol part 2]
  • [http://2012.igem.org/Team:Wageningen_UR/Protocol/RoundupCCMV| CCMV purification protocol]

Results

D25HisCCMV.PNG

background CCMV

This part is made from the wild type CCMV coat protein. CCMV is the abbreviation of Cowpea Chlorotic Mottle Virus, and is a well studied black eyed pea infecting virus. It is a virus in the family Bromoviridae, and it is one of the most studied plant viruses. As from the discovery of the virus in 1967, it was an interesting virus due to the easy cultivation and harvesting procedures involved. Interest in this virus is especially in the ability to disassemble the virus, remove the content and refold the empty particle simply by altering the pH. This makes this virus one of the first and best studied Virus Like Particles.


Besides having the characteristics of the wild type CCMV coat protein, a HIStag is added to it. This means purification with a nickel column is possible to use to purify the VLPs. (Untested)


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]

Functional Parameters

The CCMV coat protein with the first 25 amino acids replaced with a Histidine tag (6x) VLP is able to withstand temperatures up to 40 degrees Celcius, simulating a human with a mild fever. For storage it is sensible to put it at 4 degree Celcius.


CCMV VLPs are renown for its wide pH stability. It is stable between a pH range of 4.7 and 6.5. At a pH of 4.7 the CCMV is in it's unswollen form with a radius of around 14 nm. At a pH of 6.5 it is in it's swollen form, the VLPs radius is around 15-16nm. It is possible to change form, but change the pH gradually with the use of dialysis. If you change the pH too sudden, the VLPs are falling apart and forms aggregates.


Under perfect conditions (pH at 4.7, T = 4 degrees Celcius) it is possbile to store the CCMV coat protein with the first 25 amino acids replaced with a Histidine tag (6x) for a good few weeks. Be aware that aggregate formation may occure when the VLPs are exposed to changing pH or temperature.


*Safety notice*

This part is isolated from a virus and, when assembled, will form particles that very much resemble this virus in size and shape. However, no additional viral information is stored in this part and viral infection and/or replication can therefore be ruled out. It is completely safe for use in normal laboratory circumstances.

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