Part:BBa_K5403018

Fusion protein for porinMspA-based membrane anchoring of GFP

This part is a fusion protein that consists of the mycobacterial protein porinMspA (PDB ‘1UUN’, 2004), a flexible linker (BBa_K5403002) and GFP (BBa_K5403997). The mycobacterial porin MspA was shown to be part of the vesicles of M. smegmatis (Prados-Rosales et al., 2011). The protein is documented in the mycobacterial database MyCoBrowser (MyCoBrowser, MSMEG_4225). PorinMspA is an assembly of several identical domains, see figure 1. To construct this fusion protein, one domain was extended at its C-terminus. The aim being to have the GFP localized on the membrane, the fusion protein should assemble with other porin-MspA domains and reside in the membrane. Two other porinMspA-based fusion proteins with alterations in the sequence were designed (BBa_K5403017 & BBa_K5403018).

Figure 1: Structure of PorinMspA (PDB ‘1UUN’, 2004)

For cloning and expression, the plasmid pCHERRY3 can be used. This shuttle plasmid is designed such that it can be multiplied in standard laboratory E coli competent cells such as TOP10 or dH5α, and subsequently used for expression in M. smegmatis. pCHERRY3 was a gift from Tanya Parish (Addgene plasmid # 24659 ; http://n2t.net/addgene:24659 ; RRID:Addgene_24659).

References

Addgene plasmid # 24659. Sensitive detection of gene expression in mycobacteria under replicating and non-replicating conditions using optimized far-red reporters. Carroll P, Schreuder LJ, Muwanguzi-Karugaba J, Wiles S, Robertson BD, Ripoll J, Ward TH, Bancroft GJ, Schaible UE, Parish T. PLoS One. 2010 . 5(3):e9823. 10.1371/journal.pone.0009823 PubMed 20352111 Prados-Rosales, R., Baena, A., Martinez, L. R., Luque-Garcia, J., Kalscheuer, R., Veeraraghavan, U., Camara, C., Nosanchuk, J. D., Besra, G. S., Chen, B., Jimenez, J., Glatman-Freedman, A., Jacobs, W. R., Porcelli, S. A., & Casadevall, A. (2011). Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice. Journal Of Clinical Investigation, 121(4), 1471–1483. https://doi.org/10.1172/jci44261 RCSB (2004) PDB - 1UUN: Main porin from Mycobacterium smegmatis (MspA). https://www.rcsb.org/structure/1UUN

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 997
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 457
  Illegal AgeI site found at 511
  
• 1000
  COMPATIBLE WITH RFC[1000]