Fusion Protein of S-Layer SbpA and mCerulean
Fusion protein of S-layer SgsE and mCerulean.
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 (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. It is also possible to use the characteristic of mCerulean as a pH indicator or as FRET donor (Kainz et al., 2010).
|Expression (E. coli)||Localisation||Inclusion body|
|Compatibility||E. coli KRX and BL21(DE3)|
|Induction of expression||Expression of T7 polymerase + IPTG, lactose|
|Specific growth rate (un-/induced)||0.116 h-1 / 0.092 h-1|
|Doubling time (un-/induced)||5.95 h / 7.51 h|
|Purification||Molecular weight||136.9 kDa|
|Excitation / emission||435 / 477 nm|
|Immobilization behaviour||Immobilization time||4 h|
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 104
Illegal BglII site found at 221
Illegal XhoI site found at 1996
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 76
Illegal AgeI site found at 3913
- 1000Illegal BsaI.rc site found at 493
Illegal BsaI.rc site found at 622
Expression in E. coli
The SbpA|mCerulean fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0.1 % L-rhamnose and 1 mM IPTG using the autinduction protocol by Promega.
Expression of S-layer genes in E. coli
- Chassis: Promega's E. coli KRX
- Medium: LB medium supplemented with 20 mg L-1 chloramphenicol
- For autoinduction: Cultivations in LB-medium were supplemented with 0.1 % L-rhamnose and 1 mM IPTG as inducer and 0.05 % glucose
Measuring of mCerulean
- Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination
- Freeze biological samples at -80 °C for storage, keep cell-free at 4 °C in the dark
- To measure the samples thaw at room temperature and fill 200 µL of each sample in one well of a black, flat bottom 96 well microtiter plate (perform at least a repeat determination)
- Measure the fluorescence in a platereader (we used a Tecan Infinite® M200 platereader) with following settings:
- 20 sec orbital shaking (1 mm amplitude with a frequency of 87.6 rpm)
- Measurement mode: Top
- Excitation: 433 nm
- Emission: 475 nm
- Number of reads: 25
- Manual gain: 100
- Integration time: 20 µs
Kainz B, Steiner K, Möller M, Pum D, Schäffer C, Sleytr UB, Toca-Herrera JL (2010) Absorption, Steady-State Fluorescence, Fluorescence Lifetime, and 2D Self-Assembly Properties of Engineered Fluorescent S-Layer Fusion Proteins of Geobacillus stearothermophilus NRS 2004/3a, Biomacromolecules 11(1):207-214.
Sleytr UB, Huber C, Ilk N, Pum D, Schuster B, Egelseer EM (2007) S-layers as a tool kit for nanobiotechnological applications, FEMS Microbiol Lett 267(2):131-144.