Part:BBa_K525311

Fusion Protein of S-Layer SgsE and Firefly-Luciferase

Fusion protein of S-layer SgsE and firefly luciferase.

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 S-layer fusion protein is used to characterize purification methods and immobilization behaviour of enzymes fused to an S-layer.

Important parameters

Experiment	Characteristic	Result
Expression (E. coli)	Localisation	Some soluble, mostly inclusion bodies
	Compatibility	E. coli KRX and BL21(DE3)
	Induction of expression	expression of T7 polymerase + IPTG or lactose
Purification	Molecular weight	143.8 kDa
	Theoretical pI	6.02
	Reporter	Luminescence
Immobilization behaviour	Immobilization time	4 h
	Materials binding BBa_K525311	Silicon dioxide, polyethylene sulfone

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 167
  Illegal BglII site found at 1022
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 76
  Illegal AgeI site found at 4054
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1657
  Illegal SapI.rc site found at 3211
  

Expression in E. coli

The SgsE gene under the control of a T7 / lac promoter (BBa_K525303) was fused to firefly luciferase (BBa_K525999) using Freiburg BioBrick assembly for characterization experiments.

The SgsE|luciferase fusion protein was overexpressed in E. coli KRX after induction of T7 polymerase with 0.2 % L-rhamnose and induction of lac operator with 1 mM IPTG.

The following cultivation was carried out in a [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with Bioengineering DCU and software. A sequencer which automatically pumped an inducer solution after 4 h cultivation time to start protein expression was implemented. Other parameters were:

• Medium: [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_medium HSG medium] with 20 mg L^-1 chloramphenicol
• Culture volume: 2.5 L
• Starting OD₆₀₀: 0.4
• DO: 60 % airsaturation (controlled with stirrer cascade starting with 200 rpm)
• pH: 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH)
• Antifoam: BASF pluronic PE-8100
• Induction solution: 0.2 % L-rhamnose and 1 mM IPTG

The following figure shows the expression of the SgsE | luciferase S-layer fusion protein BBa_K525311 in E. coli KRX in HSG medium with autoinduction sequencer as described above. Optical density, activity of the fused luciferase, dissolved oxygen and agitator speed are plotted against the cultivation time.

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Fig. 1: Bioreactor cultivation of E. coli KRX expressing the fusion protein SgsE :: luciferase BBa_K525311 under the control of a T7 / lac promoter. Induction with 1 mM IPTG and 0.2 % L-rhamnose after 4 h controlled by sequencer. Cultivation in [http://www.gmi-inc.com/BioEngineering-KLF-Small-Laboratory-Fermenter.html#product_desc Bioengineering KLF] bioreactor with 2.5 L [http://2011.igem.org/Team:Bielefeld-Germany/Protocols/Materials#HSG_medium HSG medium] with 20 mg L^-1 chloramphenicol starting with OD₆₀₀ = 0.4. Dissolved oxygen was regulated to 60 % airsaturation (controlled with stirrer cascade starting with 200 rpm) and the pH to 7.0 (controlled with 20 % phosphoric acid and 2 M NaOH). The dissolved oxygen sensor had to be recalibrated after about 140 min.

Purification

After the analysis of cultivations with expression of SgsE | luciferase fusion proteins, different cell fractions were analyzed. It could be seen that the proteins form inclusion bodies in E. coli but that there are some soluble proteins left. This has the advantage that the proteins carrying an enzyme as fusion proteins do not have to be treated with denaturating agents like urea which destroys the enzyme (data not shown).

To capture the protein from the cell lysate an ion exchange chromatography (IEX) was carried out (binding with pH 7.0, 25 mM NaCl, quaternary amine beads, elution with 100 mM NaCl). A lot of protein was found in the flow-through. When concentrating and rebuffering the proteins with PES (polyethylene sulfone) membranes a lot of protein was lost. The S-layer proteins stuck to the membrane. Some could be removed again from the membrane after cutting out the filter and incubate it in ddH₂O over-night. This problem has to be kept in mind when using this S-layer.

The results of the purification approach are shown in Fig. 2:

Fig. 2: Luminescence corrected with the volume of the collected fraction plotted against the fraction during purification of BBa_K525311. Abbreviations: s.n. = supernatant, F = filtrate, R = retentate, FT = flow through, E10 = elution with 100 mM NaCl.

The purification strategy has to be improved. The inclusion bodies cannot be purified because urea damages the luciferase irreversible (data not shown). The loss due to adsorption of the SgsE | luciferase fusion protein to PES membranes could be avoided by using different membranes. The binding conditions of the IEX have to be improved as well. Anyway, the idea behind this purification strategy could be a starting point for a better strategy. Possibilities for improvement are:

• Different membranes for ultra- / diafiltration
• Other binding conditions for the IEX capturing step (higher pH)
• Hydrophobic interaction chromatography as purification step after IEX (works in general, data not shown)
• Size exclusion chromatography (SEC) for polishing

Stability

To test whether the S-layer protein enhances the half-life of the firefly luciferase, immobilization experiments were carried out. After the IEX described above, the elution fraction was immobilized on silicon dioxide beads or just diluted with HBSS buffer which is used for the immobilization / recrystallization of SgsE S-layer proteins. The immobilization is carried out at room temperature for 4 h. It could be seen that the luciferase activity nearly expired during this time (in the positive and the negative control). So the S-layer SgsE could not stabilize the luciferase at room temperature. The results of this experiment is shown in the figure below:

Fig. 3: Results of immobilization experiments with BBa_K525311. The stability of the luciferase activity at room temperature is decreasing quickly.