Part:BBa_K525234

Fusion Protein of mRFP, S-layer cspB from Corynebacterium halotolerans with TAT-sequence, PT7, RBS

Fusion protein of mRFP and S-layer CspB (PS2) from Corynebacterium halotolerans with TAT-sequence, T7 promoter and RBS.

S-layers (crystalline bacterial surface layer) are crystal-like layers consisting of multiple protein monomers and can be found in various (archae-)bacteria. They constitute the outermost part of the cell wall. Especially their ability for self-assembly into distinct geometries is of scientific interest. At phase boundaries, in solutions and on a variety of surfaces they form different lattice structures. The geometry and arrangement is determined by the C-terminal self assembly-domain, which is specific for each S-layer protein. The most common lattice geometries are oblique, square and hexagonal. By modifying the characteristics of the S-layer through combination with functional groups and protein domains as well as their defined position and orientation to eachother (determined by the S-layer geometry) it is possible to realize various practical applications ([http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2006.00573.x/full Sleytr et al., 2007]).

Usage and Biology

S-layer proteins can be used as scaffold for nanobiotechnological applications and devices by e.g. fusing the S-layer's self-assembly domain to other functional protein domains. It is possible to coat surfaces and liposomes with S-layers. A big advantage of S-layers: after expressing in E. coli and purification, the nanobiotechnological system is cell-free. This enhances the biological security of a device.

This fluorescent S-layer fusion protein is used to characterize purification methods and the S-layer's ability to self-assemble on surfaces.

Important parameters

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	Inclusion body
	Compatibility	E. coli KRX and BL21(DE3)
	Inductor for expression	T7 polymerase
Purification	Molecular weight	79.2 kDa
	Theoretical pI	4.54
	Excitation / emission	584 / 607 nm

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 799
  Illegal PstI site found at 1125
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 799
  Illegal PstI site found at 1125
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1876
  Illegal XhoI site found at 1332
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 799
  Illegal PstI site found at 1125
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 799
  Illegal PstI site found at 1125
  Illegal NgoMIV site found at 999
  Illegal NgoMIV site found at 2088
  Illegal AgeI site found at 87
  Illegal AgeI site found at 680
  Illegal AgeI site found at 792
  Illegal AgeI site found at 990
  Illegal AgeI site found at 1231
  Illegal AgeI site found at 1278
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1680
  Illegal BsaI.rc site found at 987
  Illegal BsaI.rc site found at 1365
  Illegal BsaI.rc site found at 1767