Part:BBa_K525121

S-layer cspB from Corynebacterium glutamicum with TAT-Sequence and lipid anchor, PT7 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)
	Compatibility	E. coli KRX
	Inductor	L-rhamnose
	Optimal temperature	37 °C
	Specific growth rate (un-/induced)	0.260 h^-1 / 0.106 h^-1
	Doubling time (un-/induced)	2,67 h / 6.52 h
Characterisation
	Number of amino acids
	Molecular weight
	Theoretical pI
	Localisation	cell membrane

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1421
Illegal XhoI site found at 248
Illegal XhoI site found at 866
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1400
Illegal SapI site found at 647
Illegal SapI site found at 859
Illegal SapI site found at 1407

Expression in E. coli

For characterization the CspB gen was fused with a monomeric RFP (BBa_E1010) using Gibson assembly.

The CspB|mRFP fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase by supplementation of 0,1 % L-rhamnose using the autinduction protocol from promega.

Figure 1: Growthcurve of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion. Cultivations were carried out in autoinduction medium at 37 ˚C.

Figure 2: RFU to OD₆₀₀ ratio of E. coli KRX expressing the fusion protein of CspB and mRFP with and without induction. A curve depicting KRX wildtype is shown for comparsion. Cultivations were carried out in autoinduction medium at 37 ˚C.

Identification and localisation

After a cultivation time of 18 h the CspB|mRFP fusion protein has to be localized in E. coli KRX. Therefore a part of the produced biomass was mechanically disrupted and the resulting lysate was washed with ddH₂O. The periplasm was detached by using a osmotic shock from another part of the cells. The existance of fluorescene in the periplasm fraction, showed in fig. 3, indicates that Brevibacterium flavum TAT-signal sequence is at least in part functional in E. coli KRX.

The S-layer fusion protein could not be found in the polyacrylamide gel after a SDS-PAGE of the lysate and the cell depris were still red. This indicated that the fusion protein intigrates into the cell membrane with its lipid anchor. For testing this assumption the washed lysate was treted with ionic, nonionic and zwitterionic detergents to release the CspB|mRFP out of the membranes.

The existance of flourescence in the detergent fractions and the proportionally low fluorescence in the wash fraction confirm the hypothesis an insertion into the cell membrane (fig. 3). An insertion of these S-layer proteins might stabilize the membrane structure and increase the stability of cells against mechanical and chemical treatment. A stabilization of E. coli expressing S-lyer proteins was discribed by Lederer et al., (2010).

An other important fact is, that there is actually mRFP fluorescence measurable in such high concentrated detergent solutions. The S-layer seems to stabilize the biologically active conformation of mRFP. The MALDI-TOF analysis of the relevant size range in the polyacrylamid gel approved the existance of the intact fusion protein in all detergent fractions (fig x).

Figure 3: Fluorescence per OD₆₀₀ progression of the CspB/mRFP (BBa_E1010) fusion protein initiating with the cultivation fractions up to the detergent fractions of the seperate denaturations. Cultivations were carried out in autoinduction medium at 37 ˚C. The cells were mechanically disrupted and the resulting biomass was wahed with ddH₂O and resuspendet in the respective detergent. The used detergent acronyms stand for: SDS = 10 % (v/v) sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % (v/v) n-lauroyl sarcosine; CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

MALDI TOF analysis was first used to identify the location of the fusion protein in different fractions. Fractions of media supernatant after cultivation, periplasmatic isolation, cell lysis, denaturation in 6 M urea and the following wash with 2 % (v/v) Triton X-100, 2 % SDS (w/v) were loaded onto a SDS-PAGE and fragments of the gele were measured with MALDI TOF.

Figure 4: SDS-PAGE of CspB/mRFP (BBa_E1010) fusion protein. Lanes are fractions of media (M), periplasmatic isolation (PP), cell lysis (L), denaturation (D) and wash with Triton X-100 (T). Used Marker is PageRuler ^TM Prestained Protein Ladder SM0671. Marked regions were cut out and prepared for MALDI TOF analysis.

The following table shows the sequence coverage (in %) of our measurable gele samples with the amino acid sequence of fusion protein CspB/mRFP (BBa_E1010).