Difference between revisions of "Part:BBa K525123"

(→‎Identification and localisation)
(→‎Identification and localisation)
Line 67: Line 67:
 
The existance of flourescence in the detergent fractions and the non-existancing fluorescence in the wash fraction confirm the hypothesis of 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).
 
The existance of flourescence in the detergent fractions and the non-existancing fluorescence in the wash fraction confirm the hypothesis of 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.
+
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 4).  
  
 
In comparison with the mRFP fusion protein of [https://parts.igem.org/Part:BBa_K525121 K525121], wich has a TAT-sequence, a minor relative fluorescence in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD<sub>600</sub> after 12 h of cultivation (fig. 2) indicates this rusults in a postive effect of the TAT-sequence on the protein stability.
 
In comparison with the mRFP fusion protein of [https://parts.igem.org/Part:BBa_K525121 K525121], wich has a TAT-sequence, a minor relative fluorescence in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD<sub>600</sub> after 12 h of cultivation (fig. 2) indicates this rusults in a postive effect of the TAT-sequence on the protein stability.

Revision as of 00:39, 22 September 2011

S-layer cspB from Corynebacterium glutamicum with lipid anchor and PT7 and RBS

Bielefeld-Germany2011-S-Layer-Geometrien.jpg

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.

The S-layer of C. glutamicum is characterized by a hexagonal lattice symmetry. Attachment between S-layer and cell wall was found to be due to the hydrophobic carboxy-terminus of the PS2 protein [http://www.sciencedirect.com/science/article/pii/S016816560400241X Hansmeier et al., 2004]).

Important parameters

Experiment Characteristic Result
Expression (E. coli) Localisation cell membrane
Compatibility E. coli KRX
Inductor for expression L-rhamnose for induction of T7 polymerase
Specific growth rate (un-/induced) 0.245 h-1 / 0.093 h-1
Doubling time (un-/induced) 2.82 h / 7.42 h

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 1334
    Illegal XhoI site found at 161
    Illegal XhoI site found at 779
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1313
    Illegal SapI site found at 560
    Illegal SapI site found at 772
    Illegal SapI site found at 1320


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, cultivated at 37 °C in autoinduction medium with, respectively, without inductor. A curve depicting KRX wildtype is shown for comparsion. After induction at approximately 6 h the OD600 of the induced K525123 visibly drops when compared to the uninduced culture. While the induced culture grow significantly slower than KRX wildtype the uninduced seems to be unaffected.
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. After induction at approximately 6 h the RFU to OD600 ratio starts to rise in the induced culture. Compared to the uninduced culture the ratio is roughly eight times higher. Most likely due to basal transcription the RFU to OD600 ratio of the uninduced culture starts to rise after 12 hours. The KRX wildtype shows no variation in the RFU to OD600 ratio.

Identification and localisation

After a cultivation time of 18 h the mRFP|CspB fusion protein has to be localized in E. coli KRX. Therefor a part of the produced biomass was mechanically disrupted and the resulting lysate was wahed with ddH2O. From the other part the periplasm was detached by using a osmotic shock.

The S-layer fusion protein could not be found in the polyacrylamide gel after a SDS-PAGE of the lysate. 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 mRFP|CspB out of the membranes.

The existance of flourescence in the detergent fractions and the non-existancing fluorescence in the wash fraction confirm the hypothesis of 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 4).

In comparison with the mRFP fusion protein of K525121, wich has a TAT-sequence, a minor relative fluorescence in all cultivation and detergent fractions was detected (fig. 3). Together with the decreasing RFU/OD600 after 12 h of cultivation (fig. 2) indicates this rusults in a postive effect of the TAT-sequence on the protein stability.

Figure 3: Fluorescence progression of the mRFP(BBa_E1010)/CspB 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 % sodium dodecyl sulfate; UTU = 7 M urea and 3 M thiourea; U = 10 M urea; NLS = 10 % n-lauroyl sarcosine; 2 % CHAPS = 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

MALDI TOF analysis was used to identify the location of the fusion protein in different fractions. Fractions of medium supernatant after cultivation (M), periplasmatic isolation (PP), cell lysis (L) and the following wash with ddH2O, samples were loaded onto a SDS-PAGE. After comparison with same treated fraction of E. coli KRX all gel bands in a defined size area were cutted out of the gel and analysed with MALDI TOF. Results are shown in Fig. 4.


Figure 4: MALDI TOF measurement of the mRFP(BBa_E1010)/CspB fusion protein. Data is shown in a SDS-PAGE. Colours show the sequence coverage of MALDI TOF measurement. On the left side of the gel samples fractions of the fusion protein are shown, on the right side of the gel fractions of an equally treated E. coli KRX are shown. Measurement was performed with a ultrafleXtremeTM by Bruker Daltonics using the software FlexAnalysis, Biotools and SequenceEditor.

Results show that the fusion protein of mRFP(BBa_E1010)/CspB without Tat sequence and with lipid anchor has only been identified in the lysis fraction. However, in conclusion with absent Tat sequence, the protein has not been identified in the periplasm.

The influence of other detergents to desintegrate the S-layer fusion protein was tested after disputing the cells with a ribolyser. The cell pellet was incubated in 10 % (v/v) Sodium dodecyl sulfate (SDS), in 7 M urea and 3 M thiourea (UTU), in 10 M urea (U) in 10 % (v/v) n-lauroyl sarcosine (NLS) and in 2 % CHAPS (C). Samples of the incubations with these detergents were loaded onto a SDS-PAGE prior to measurement with MALDI TOF (Fig. 6).


Figure 6: Influence of diffent detergents on the desintegration of the fusionprotein CspB/mRFP (BBa_E1010) from the cell membrane of E. coli KRX. The coloured Marker shows the sequence coverage of MALDI TOF measurement. Abbreviations are: SDS (Sodium dodecyl sulfate 10 % (w/v)), NLS ((10 % (w/v) m-lauroyl sarcosine), UTU (3 M thiourea, 7 M urea), U (10 M urea), CH (2% CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), Ma Marker (PageRuler TM Prestained Protein Ladder SM0671).On the left half of the gel fractions of the S-layer fusion protein are displayed, on the right half fractions of E. coli KRX are displayed.

The results of the MALDI TOF measurement clearly demonstrates, that all used detergents are applicable to desintegrate the S-layer fusion proteins from the bacterial cell membrane of E. coli. Fluorescence measurement of fractions, treated with the detergents, show significantly different values, indicating that some of the detergents (e.g. 3 M thiourea, 7 M urea) have a strong effect on protein folding. The samples taken from gel lanes of E. coli KRX show no sequence coverage, therefore not similar proteins are naturally induced in E. coli.