As all the other effector proteins we performed a first analytical preparation in a 2 L LB-media scale in order to learn about the protein and then up-scaled the production to 6 x 2 L of LB media. The production was carried out cytoplasmatically in ''E. coli'' BL21. The culture was grown to OD 0.8 was induced using 5 mM of arabinose and the protein production was carried out for 5 hours. Cell disruption was performed using ultrasonic sound and subsequently the cell lysate was dialyzed against 1 x SA-Buffer that contained additionally 5 mM of the milde oxidation reagent potassium ferrocyanide in order to form the disulfide bridges present in the laccase structure (see previous paragraph).<br>
As all the other effector proteins we performed a first analytical preparation in a 2 L LB-media scale in order to learn about the protein and then up-scaled the production to 6 x 2 L of LB media. The production was carried out cytoplasmatically in ''E. coli'' BL21. The culture was grown to OD 0.8 was induced using 5 mM of arabinose and the protein production was carried out for 5 hours. Cell disruption was performed using ultrasonic sound and subsequently the cell lysate was dialyzed against 1 x SA-Buffer that contained additionally 5 mM of the milde oxidation reagent potassium ferrocyanide in order to form the disulfide bridges present in the laccase structure (see previous paragraph).<br>
-
The Streptavidin affinity chromatography yielded a satisfying elution peak that was then concentrated using a centrifugal filter unit (MWCO: 30 kDa). The concentrated protein was then applied to a ÄKTA purifier equipped with a Superdex 200 10/30 size exclusion chromatography column.
+
The Streptavidin affinity chromatography yielded a satisfying elution peak that was then concentrated using a centrifugal filter unit (MWCO: 30 kDa). The concentrated protein was then applied to a ÄKTA purifier equipped with a Superdex 200 10/30 size exclusion chromatography column. The chromatogram shows three major peaks with a first peak near the void volume that corresponds to aggregated protein >600 kDa, a second elution peak of high molecular protein and a third elution peak corresponding to the desired laccase protein.
For the development of a transgenic water filter it is an essential task to create a collection of well described and functional effector proteins which are either able to bind (BioAccumulation) or to degrade (BioDegradation) xenobiotics present in the aquatic environment. This task was completed by the production of relevant effector proteins in E. coli and to complete their subsequent purification and characterization. Deliberately we have chosen some well established BioBricks from the last years such as a laccase (<partinfo>BBa_K1159002</partinfo>) or the catechol dioxigenase (<partinfo>BBa_K648011</partinfo>) to improve these BioBricks. Beside these improvements we also added new BioBricks to the registry which we characterized in vitro such as the erythromycin esterase (EreB) (<partinfo>BBa_K1159000</partinfo>) or the NanoLuc luciferase (<partinfo>BBa_K1159001</partinfo>) which will be a useful tool for subsequent generations of iGEM students. For technical questions on our experiments, please see protein biochemical methods for further information. Beside these experiments with recombinant proteins we also characterized our stable transformed moss strains (see GM-Moss section).
The erythromycin esterase (EreB) is an enzyme found in some strains of Escherichia coli and which was introduced to the Parts Registry by the TU Munich iGEM Team 2013 in RFC[25]. As this BioBrick is derived from Escherichia coli it was to expect that it is well expressing in our experiments on recombinant effector proteins. For further information on the theoretical background of EreB please see our BioDegradation page.
Protein Data Table for the erythromycin esterase (EreB) <partinfo>BBa_K1159000</partinfo>
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCAGGTTCGAA ... GTTTATGAAACCGGT ORF from nucleotide position -8 to 1260 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
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Production and purification of recombinant EreB
Figure A: Streptavidin affinity chromatography for the erythromycin esterase
Figure B: Analytical size exclusion chromatography on a Superdex 200 10/30 column
Figure C: Preparative size exclusion chromatography on a Superdex 75 16/60 column
The recombinant production and purification was carried out twice, in a first attempt 2 L of LB-media were used for an analytical purpose whereas in the second attempt we produced enough purified enzyme for all subsequent experiments. This preparation was carried out in 6 x 2L of LB media. Protein production was in both cases induced at OD = 0.8 by adjusting the cell culture to 5 mM of arabinose and was carried out for 4 h for the first and 5 h for the second preparation. Cell disruption was performed by ultrasonic sound in both cases. The cell lysate was then dialyzed against 5 L of SA-buffer and subsequently applied to streptavidin affinity columns. After the application of the protein, the column was washed with SA-buffer until a base line was reached. Afterwards the protein was eluted using 5 mM biotin. During the first preparation 2-mercapto-ethanol was added after the chromatographic steps. In order to avoid oxidation of cysteine residues to disulphid-bridges, which is not desired for the cytosolic EreB protein, the preparative purification was carried out with buffers containing 5 mM of 2-mercapto-ethanol in all buffers. When comparing the size exclusion chromatograms, obtained from the analytical and the preparative purification, it can be stated that there is still a considerable aggregation peak near the void volume (Fig. B) of the column in the first attempt, which was nearly not the case for the preparative preparation (Fig. C). Therefore we would give the advise to use strictly reducing conditions while working with recombinant EreB. The finally resulting yields of the preparative purification have been determined by absorption measurement of the aromatic amino acids at 280. The total yield was determined to 25 mg of pure protein which is 2.1 mg/L of LB culture.
Kirby-Bauer Assay: Measuring remaining erythromycin on a pertri dish
Figure 2 Kirby-Bauer assay of recombinant EreB incubated with erythromycin:
The Kirby-Bauer assay is an agar diffusion test, with which it is very easy to examine the decomposition activity of the enzyme. We also analyzed the decomposition activity of the recombinant EreB by LC-MS. The recombinant protein is incubated with the antibiotic and the reaction is stopped with methanol. To be sure that the enzyme is inhibited and the reaction does not go on, we additionally heated the reaction mixture (see table 2) after stopping for two minutes at 50°C and then shock froze the mixture in liquid nitrogen. The effect of methanol, heating and shock freezing on the bacterial strain and the substrate were checked before and have no influence.
Then bacteria from a quit dense liquid culture are plated on a LB agar plate without antibiotics under sterile conditions and spread with sterile cotton tip applicators. We used the bacterial strain Micrococcus luteus which was generously donated from the [http://www.micbio.wzw.tum.de/cms/docs/scherer-anzeigen.php chair of microbiology by Prof. Scherer`s group] and is mentioned to be specifically sensitive to Erithromycin http://pubs.acs.org/doi/abs/10.1021/bi201790u?mi=0&af=R&pr... Wright et al., 2012. Now several 6 mm filter paper discs are placed on the bacterial lawn in adequate intervals and 8 µl of the spinned down reaction mixture is added onto one disc. The mixture diffuses from the filter paper into the agar. The concentration of the compound will be highest next to the disk, and will decrease with increasing distance. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is called the zone of inhibition.
table 2: Reaction mixture
substance
amount
stock solution
EreB recombinant protein 40 nM
0.18 µl
11 µM EreB in PBS with 10 mM ß-mercaptoethanol, 2% glycerol (v/v) and 300 mM NaCl
Erythromycin 0.36 mM
3 µl
Erythromycin in ethanol, 6 mM
Tris-HCl Buffer pH 7.5 100 mM
10 µl
Tris-HCl Buffer pH 7.5, 500 mM
NaCl 0.08 M
4 µl
NaCl in water, 1 M
ddH20
32.82 µl
ddH20
TOTAL:
50 µl
As negative controls there is one mixture containing no substrate or enzyme and two mixtures without the enzyme but with antibiotic, which were incubated for 0 minutes and 6 hours.
As expected the assay shows a gradually decreasing zone of inhibtion with increasing reaction time. In the first half hour the zone is more or less constant although the enzyme is constantly degrading the antibiotic. This can be explained by the fact that the mixture only diffuses a certain range into the agar and in the first few time steps of the reaction the concentration of the antibiotic within this range is above the http://en.wikipedia.org/wiki/Minimum_inhibitory_concentration minimum inhibitory concentration (MIC), so the bacteria is inhibited independently of the exact concentration. After 3 hours all Erythromycin was degraded so there is no zone of inhibition anymore. Also we can see that the antibiotic does not degrade by itself significantly over 6 hours since the zones of inhibition in both negative controls are pretty much the same.
Characterisation of the enzyme
Figure 3: Reaction mechanism of EreB hydrolyzing the model ester p-NPB into a chromogenic product
To characterize the enzyme we performed a spectrophotometric assay using the model ester p-nitrophenyl-butyrate (p-NPB) as a substrate for EreB. The chromogenic product p-Nitrophenyl shows characteristic absorbance at 405 nm. The reaction takes place even faster than hydrolysis of erythromycin since p-NPB is already highly activated and therefore readily hydrolyzed. This is also the reason why it degrades quite rapidly by itself so reactions have to be measured soon after being set up and taking a negative control is especially important.
Kinetics
Figure 3:
PH optimum
Figure 4: Reaction of EreB with p-NPB in different pH buffers
Ionic strength optimum
Figure 5: Reaction of EreB with p-NPB at different salt concentrations
Laccase
Laccases are an important group of effector proteins that can be utilized in bioremediation to degrade xenobiotics. They are copper-containing enzymes which catalyze the oxidation of a wide range of substances. It was interesting for us as the two important xenobiotics ethinylestradiol and diclofenac can be degraded using laccases and the general function can easily be investigated using the commonly used substrate ABTS.
During the planing phase of our project we decided not to add a new laccase to the Parts Registry but instead to continue the work on the best availible laccase. The iGEM Bielefeld Germany has created BioBricks for five different laccases during the last summer. So we contacted this iGEM team and got the advise that the laccase derived from Bacillus pumilus <partinfo>BBa_K863000</partinfo> seems the best choice for our effector collection. As we wanted to try this effector protein in different localisations such as cytoplasmatic, secreted or receptor bound we converted this enzyme to RFC[25] in order to make it compatible for protein fusions.
Protein Data Table for the Laccase from Bacillus pumilus <partinfo>BBa_K863000</partinfo>
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCAACCTAGAA ... GATATCATCACCGGT ORF from nucleotide position -8 to 1530 (excluding stop-codon)
Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)
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Laccase is a secreted enzyme
Figure 6:
For the production of effector proteins it is allwasy essential to know whether they are naturally cytoplasmatic or whether they are secreted. Cytoplasmatic proteins usually do contain a higher portion of cysteine residues as they are a normal amino acid residue as no disulfide bridges can be formed under the reducing conditions of the cytoplasm. In contrast secreted protein often do contain disulfide briges which make them more stable to environmental stress. The disulfide formation is possible for the secreted proteins as the oxidizing conditions facilitate the bridge formation. On the other hand there is a selection pressure for secreted proteins not to contain exposed cysteine residues that are not involved in the formation of disulfide bridges. Such free cysteine residues would leed to aggregation of proteins which would probably hamper their activity.
Thus it was an important point to find out whether the used laccase is a secreted enzyme as this would increase the chance to express it in a functional form on the extracellular part of our moss and on the other hand it was also an important information for the production of recombinant protein. To solve this question we did an alignment of the laccase BioBrick and the closest related solved structure. The sturcure contains disulfid bridges for example Cys229-Cys322. The comparison of the position of these residues within the amino acid sequece alignment shows that these residues are conserved and it can thus be assumed that the laccase BioBrick we used also contains disulfid bridges that have to be formed under oxidizing conditions and will increase the stability of our effector protein.
We thus used 10 mM of potassium ferrocyanide which is a milde oxidisation reagent subsequent to the cell disruption and did not use any 2-mercapto ethanol as a reducing agent during the protein purification.
Analytical preparation
As all the other effector proteins we performed a first analytical preparation in a 2 L LB-media scale in order to learn about the protein and then up-scaled the production to 6 x 2 L of LB media. The production was carried out cytoplasmatically in E. coli BL21. The culture was grown to OD 0.8 was induced using 5 mM of arabinose and the protein production was carried out for 5 hours. Cell disruption was performed using ultrasonic sound and subsequently the cell lysate was dialyzed against 1 x SA-Buffer that contained additionally 5 mM of the milde oxidation reagent potassium ferrocyanide in order to form the disulfide bridges present in the laccase structure (see previous paragraph).
The Streptavidin affinity chromatography yielded a satisfying elution peak that was then concentrated using a centrifugal filter unit (MWCO: 30 kDa). The concentrated protein was then applied to a ÄKTA purifier equipped with a Superdex 200 10/30 size exclusion chromatography column. The chromatogram shows three major peaks with a first peak near the void volume that corresponds to aggregated protein >600 kDa, a second elution peak of high molecular protein and a third elution peak corresponding to the desired laccase protein.
Figure 8:
Figure 9: Streptavidin affinity chromatography for the laccase <partinfo>BBa_K863000</partinfo>
Structural consideration of Laccase from B. pumilus
The enzymatic activity of the purified laccae was determined by the ABTS-assay. In a first pre experiment the appropriate dilution factor was determined to 100-fold. The elution fractions obtained from size exclusion chromatography were diluted 1:100 in PBS and in an ELISA plate 100 µl of the enzyme and 100 µl of ABTS substrate were mixed and a kinetic measurement at 405 nm was performed. The absorption at 280 nm in the SEC chromatogramm (blue) identifies three main protein peaks, with a first peak corresponding to aggregated protein, a shoulder which also corresponds to higher molecular protein and a single peak which was proposed to be the monomeric laccase. The relative activity obtained for the different elution fractions was plotted in the same diagramm and shows a clear peak which matches the laccase peak in the SEC. Beside this major peak a second smaller peak of active fraction was visible which appeared in earlier elution fractions and might correspond to dimerized laccase. As the laccase is a secreted enzyme which also bears disluphide bonds it was produced in the cytoplasm and subsequently it was oxidized to form the proper disulphide bond. As this process might be only partial there is a possiblity for the formation of disulphid dimers. Never the less the fractions 14 to 17 were pooled for further experiments as they showed the highest enzymatic activity. The protein concentration of the pooled fraction was determined to 0.48 mg/ml after SEC.
Characterization of the enzyme
Figure 12:
Figure 12: Reaction of Laccase with ABTS in different pH buffers
Figure 13: Reaction of Laccase with ABTS at different salt concentrations
The Nano Luciferase (NanoLuc) which was introduced in 2013 by Promega is a new member of the luciferase reporter gene/protein familiy and shows some advantages compared to the other family members. The NanoLuc is very small (19 kDa) compared to the firefly luciferase (61 kDa) and the Renilla luciferase (36 kDa). On the other hand it is also said that the specific activity of the NanoLuc is about 150-fold stronger compared to conventional luciferases and the background caused by autoluminescense of the substrate shel be smaller.
Protein Data Table for the Nano Luciferase <partinfo>BBa_K1159001</partinfo>
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCGTGTTCACC ... ATCCTTGCTACCGGT ORF from nucleotide position -8 to 516 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
Subcellular Localization (reliability in brackets)
Archaea:
secreted (100%)
Bacteria:
secreted (51%)
Eukarya:
cytosol (12%)
Gene Ontology (reliability in brackets)
Molecular Function Ontology:
-
Biological Process Ontology:
-
Predicted features:
Disulfid bridges:
-
Transmembrane helices:
-
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Production in E. coli and purification
Figure 3:Analytical size exclusion chromatography on a Superdex 200 10/30 column showing a single elution peak for the NanoLuc.
Figure 3:Preperative size exclusion chromatography on a Superdex 75 10/30 column showing a single elution peak for the NanoLuc.
Therefore the NanoLuc was synthesized as a BioBrick in RFC[25] and was produced in E. coli using the pBad expression system with a C-terminal Strep-tag. After the production (2 l of LB-media for analytical and 12 l for preparative preparations) the cells were disrupted using sonification and the lysate was dialysed against 5 l of 1x SA-buffer. Afterwards the lysate was applied to a Streptavidin-Affinity (SA) column and was subsequently washed using SA-Buffer until a baseline was reached and the protein was then eluted using 5 mM of biotin (Attention: These are special columns which are not availible commercially. If you are using commercial colum material you have to use d-Desthiobiotin because usual biotin will elute your protein but you will not be able to regenerate the column after your chromatography). After the SA-chromatography the protein was concentrated using centrifugal concentration units (MWCO: 10 kDa). The concentrated protein was then applied on a Superdex S200/75 size exclusion chromatography.
There is no structure availible for the NanoLuc in the [http://www.rcsb.org/pdb/home/home.do Protein Data Bank]. In our protein modelling we used homolgy search and identified the structure [http://www.rcsb.org/pdb/explore/explore.do?structureId=3PPT 3ppt_A] as the solved structure with the highest homology to the NanoLuc which has only 21% identify with a similarity of only 0.359. The result of the homology search is shown as annimated gif in Figure xx (please see our How To for an introduction). The protein was dialysed against 1x CD-buffer and subsequently a circular dichroism spectroscopy was tanken (learn about CD spectroscopy). The CD spectrum was used to predict the secondary structure content of the NanoLuc which could be determined to 35.1% helix, 27.6% b-strand, 18.5% turn and 18.8% random. As there is only a poor homology present, a detailed comparison of the determined and the predicted secondary structure is not possible. But it can be stated that both show a balanced content of different secondary structures and that the produced protein is present in an folded conformation. The mixed secondary structure content is also in consistance with the predicted secondary structure shown in the AutoAnnotator sequence window (click on show).
Activity determination of Luminescense
The activity of the produced NanoLuc was investigated by its luminescense. The luminescense
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCAACAAAGGT ... GTGCTGACCACCGGT ORF from nucleotide position -8 to 924 (excluding stop-codon)
Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.
Nucleotide sequence in RFC 10: (underlined part encodes the protein) ATGGACTTT ... TTCCTGAGCTAGTAG ORF from nucleotide position 1 to 627 (excluding stop-codon)
Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)
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Protein phosphatase 1 (from Homo sapiens) <partinfo>BBa_K1159004</partinfo>
The production of recombinant protein phosphatase 1 was part of our collaboration with Dundee iGEM team 2013. They developed this BioBrick which naturally binds [http://de.wikipedia.org/wiki/Microcystine microcystine], an important environmental toxin.
Protein Data Table for the protein phosphatase 1 (PP1)
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCGGATCCGCG ... GCAAAGAAAACCGGT ORF from nucleotide position -8 to 978 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)
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Production and purification of recombinant PP1 protein
Figure A: Streptavidin affinity chromatography of protein phosphatase 1
Figure B: Size exclusion chromatography of protein phosphatase 1 on Superdex 200 10/30 column
We converted this BioBrick to RFC[25] and cloned it afterward into the expression vector pBad_C-terminal_Strep. Beside the recombinant characterisation we also created a transgenic moss transformed with a receptor containing PP1 in its extracellular domain. The recombinant protein production was carried out in E. coli BL-21 which was grown in 2 L of LB-media. The protein production was induced at OD = 0.8 by addition of arabinose to a concentration of 5 mM. The cells were harvested after 4 h and subsequently resuspended in 20 mL SA-buffer with 5 mM 2-mercapto ethanol. Cell disruption was performed using ultrasonic sound. The dialysed cell extracte was applied to a streptavidin affinity column which was washed until a base line was reached and was subsequently eluted using SA-buffer containing 5 mM of biotin and 5 mM of 2-mercapto ethanol. The elution peak recorded by the continuous measurement of the absorbance at 280 nm indicated a good yield of recombinant protein (Fig. A). As a second purification step after the streptavidin affinity chromatography we concentrated the protein in centrifugal filter units in order to apply it to a size exclusion chromatography. During the concentration process there were clear signal for precipitated protein which appeared as white flakes in the concentration filter unit. This effect was by far the most drastic precipitation of recombinant that was detected during this iGEM project. Anyhow the concentrated protein was centrifuged for 5 min at 13 200 RPM to remove particles of precipitated protein and was then applied to an ÄKTA purified with a Superdex 200 10/30 column (Fig. B). There were four different peaks present in the chromatogramm which most probably correspond to (1) a peak created by aggregated protein which runs in the voit volume of the size exclusion colum, (2) a diffuse peak which might correspond to multimeric PP1, (3) a sharp peak at the elution volume where the recombinant PP1 was expected and (4) a peak which is most probably caused by low molecular buffer substances. The general signal intensity obtained in the size exclusion was very low indicating low protein concentrations. The maximal peak obtained for PP1 had an absorbance of 50 milli Absorption Units whereas this was for example for the NanoLuc in the range of 1000 for a comparable experiment. The fact that this protein does not tolerate the concentration procedure gibes an indication that it is fragile and tends to aggregation when stress is applied. Finally recombinant PP1 protein was prepared although the yield was very low compared with other effector proteins.
Possible reasons for the instability of PP1 in vitro
The high aggregation and denaturation tendency of the protein phosphatase 1 which was observed in the analytical preparation motivated this search for a possible explanation. As a first hint the amino acid composition calculated by the AutoAnnotator was examined and it can be seen that 13 cysteine residues are present in the sequence of the protein phosphatase. This corresponds to 4% of the total number of amino acid residues comapred to 2.8% cystein residues in average protein. This information taken together with the molecular function of the protein phosphatase 1 clearly shows that this BioBrick is a cytoplasmatic protein which fulfills its function in the reducing milieu of the cytoplasm and tends to aggregation when exposed to an oxidizing milieu such a the periplasm of E. coli for example.
Secondly the amino acid sequence produced by the AutoAnnotator was fed into the bioinformatic tool [http://web.expasy.org/cgi-bin/protparam/protparam ProtParam which has a stability prediction for proteins. The result was: "The instability index (II) is computed to be 42.99 - This classifies the protein as unstable."
Taken both these indications into account it still seems a good idea to express the protein phosphatase 1 cytoplasmatically to bind microcystine but it is not advisable to think about a secreted version of PP1 as the protein seems to unstable for this application.
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCGTTGATACC ... GCTCATATTACCGGT ORF from nucleotide position -8 to 345 (excluding stop-codon)
Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.
http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984 Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.
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