Team:TU-Munich/Results/Recombinant

From 2013.igem.org

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===Aktivity determination using ABTS===
===Aktivity determination using ABTS===
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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 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.  
[[File:TUM13_ABTS-oxidation.png|thumb|left|250px| Figure 3:]]
[[File:TUM13_ABTS-oxidation.png|thumb|left|250px| Figure 3:]]
[[File:TUM13_Laccase_activity.png|thumb|right|320px| Figure 3:]]
[[File:TUM13_Laccase_activity.png|thumb|right|320px| Figure 3:]]

Revision as of 11:58, 27 September 2013


Characterization of recombinant effector proteins

Text. See proteinbiochemical methods for further informatons.

Table 1: Investigated Proteins
Protein BioBrick RFC Affinity tag Size [kDa] Disulphid bridges comment
Eryhtromycin esterase (EreB) <partinfo>BBa_K1159000</partinfo> RFC25 c-term. Streptag II
Laccase <partinfo>BBa_K1159002</partinfo> RFC25 c-term. Streptag II
Nano Luciferase <partinfo>BBa_K1159001</partinfo> RFC25 c-term. Streptag II
XylE <partinfo>BBa_E0040</partinfo>
PP1 <partinfo>BBa_K1159004</partinfo>
YFP_TEV_CFP <partinfo>BBa_K1159112</partinfo>


Eryhtromycin Esterase

[...] description [...] reaction


Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
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: (RFC25 scars in shown in bold, other sequence features underlined; both given below)
1 MAGRFEEWVKDKHIPFKLNHPDDNYDDFKPLRKIIGDTRVVALGENSHFIKEFFLLRHTLLRFFIEDLGFTTFAFEFGFAEGQIINNWIHGQGTDDEIGR
101 FLKHFYYPEELKTTFLWLREYNKAAKEKITFLGIDIPRNGGSYLPNMEIVHDFFRTADKEALHIIDDAFNIAKKIDYFSTSQAALNLHELTDSEKCRLTS
201 QLARVKVRLEAMAPIHIEKYGIDKYETILHYANGMIYLDYNIQAMSGFISGGGMQGDMGAKDKYMADSVLWHLKNPQSEQKVIVVAHNAHIQKTPILYDG
301 FLSCLPMGQRLKNAIGDDYMSLGITSYSGHTAALYPEVDTKYGFRVDNFQLQEPNEGSVEKAISGCGVTNSFVFFRNIPEDLQSIPNMIRFDSIYMKAEL
401 EKAFDGIFQIEKSSVSEVVYETG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
None of the supported features appeared in the sequence
Amino acid composition:
Ala (A)27 (6.4%)
Arg (R)16 (3.8%)
Asn (N)20 (4.7%)
Asp (D)29 (6.9%)
Cys (C)3 (0.7%)
Gln (Q)14 (3.3%)
Glu (E)30 (7.1%)
Gly (G)31 (7.3%)
His (H)15 (3.5%)
Ile (I)36 (8.5%)
Leu (L)34 (8.0%)
Lys (K)29 (6.9%)
Met (M)12 (2.8%)
Phe (F)31 (7.3%)
Pro (P)14 (3.3%)
Ser (S)22 (5.2%)
Thr (T)19 (4.5%)
Trp (W)4 (0.9%)
Tyr (Y)19 (4.5%)
Val (V)18 (4.3%)
Amino acid counting
Total number:423
Positively charged (Arg+Lys):45 (10.6%)
Negatively charged (Asp+Glu):59 (13.9%)
Aromatic (Phe+His+Try+Tyr):69 (16.3%)
Biochemical parameters
Atomic composition:C2204H3348N568O636S15
Molecular mass [Da]:48459.2
Theoretical pI:5.55
Extinction coefficient at 280 nm [M-1 cm-1]:50310 / 50498 (all Cys red/ox)
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.74)good (0.77)good (0.75)good (0.80)good (0.76)good (0.66)
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Analytical präparation

Figure 3:
Figure 3:
Figure 3:

Degradation of a chromogenic esterase substrate: 4-Nitrophenyl butyrate

Degradation of Erythromycin

Reaction conditions HPLC Kirby Bauer-Assay

[...] Characterization




Laccase

[...] description [...] reaction [...] production


Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
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)
1 MAGNLEKFVDELPIPEVAKPVKKNPKQTYYEIAMEEVFLKVHRDLPPTKLWTYNGSLPGPTIHANRNEKVKVKWMNKLPLKHFLPVDHTIHEGHHDEPEV
101 KTVVHLHGGVTPASSDGYPEAWFSRDFEATGPFFEREVYEYPNHQQACTLWYHDHAMALTRLNVYAGLAGFYLISDAFEKSLELPKGEYDIPLMIMDRTF
201 QEDGALFYPSRPNNTPEDSDIPDPSIVPFFCGETILVNGKVWPYLEVEPRKYRFRILNASNTRTYELHLDNDATILQIGSDGGFLPRPVHHQSFSIAPAE
301 RFDVIIDFSAYENKTITLKNKAGCGQEVNPETDANIMQFKVTRPLKGRAPKTLRPIFKPLPPLRPCRADKERTLTLTGTQDKYGRPILLLDNQFWNDPVT
401 ENPRLGSVEVWSIVNPTRGTHPIHLHLVQFRVIDRRPFDTEVYQSTGDIVYTGPNEAPPLHEQGYKDTIQAHAGEVIRIIARFVPYSGRYVWHCHILEHE
501 DYDMMRPMDIITG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 130 to 131
Amino acid composition:
Ala (A)27 (5.3%)
Arg (R)29 (5.7%)
Asn (N)23 (4.5%)
Asp (D)31 (6.0%)
Cys (C)5 (1.0%)
Gln (Q)14 (2.7%)
Glu (E)37 (7.2%)
Gly (G)30 (5.8%)
His (H)23 (4.5%)
Ile (I)31 (6.0%)
Leu (L)41 (8.0%)
Lys (K)27 (5.3%)
Met (M)10 (1.9%)
Phe (F)24 (4.7%)
Pro (P)47 (9.2%)
Ser (S)18 (3.5%)
Thr (T)33 (6.4%)
Trp (W)8 (1.6%)
Tyr (Y)22 (4.3%)
Val (V)33 (6.4%)
Amino acid counting
Total number:513
Positively charged (Arg+Lys):56 (10.9%)
Negatively charged (Asp+Glu):68 (13.3%)
Aromatic (Phe+His+Try+Tyr):77 (15.0%)
Biochemical parameters
Atomic composition:C2671H4072N718O760S15
Molecular mass [Da]:58883.0
Theoretical pI:6.03
Extinction coefficient at 280 nm [M-1 cm-1]:76780 / 77093 (all Cys red/ox)
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.72)good (0.75)good (0.68)good (0.74)good (0.77)good (0.67)
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Laccase a is a secreted enzyme

Figure 3:
--->


Analytical präparation

[...] Characterization

Figure 3:
Figure 3:

Structural consideration of Laccase from B. pumilus

File:TUM13 Animation Laccasex.gif
Figure 3:

Aktivity determination using ABTS

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.

File:TUM13 ABTS-oxidation.png
Figure 3:
Figure 3:

Oxidation of relevant xenobiotics

Diclofenac

Estradiol

Nano Luciferase

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein)
 ATGGCCGGCGTCTTCACA ... ATTCTGGCGACCGGT
 ORF from nucleotide position -8 to 516 (excluding stop-codon)
Amino acid sequence: (RFC25 scars in shown in bold, other sequence features underlined; both given below)
1 MAGVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT
101 LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILATG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
RFC25 scar (shown in bold): 163 to 164
Amino acid composition:
Ala (A)4 (2.3%)
Arg (R)7 (4.0%)
Asn (N)8 (4.6%)
Asp (D)12 (6.9%)
Cys (C)1 (0.6%)
Gln (Q)7 (4.0%)
Glu (E)8 (4.6%)
Gly (G)22 (12.6%)
His (H)4 (2.3%)
Ile (I)18 (10.3%)
Leu (L)16 (9.1%)
Lys (K)7 (4.0%)
Met (M)3 (1.7%)
Phe (F)8 (4.6%)
Pro (P)6 (3.4%)
Ser (S)6 (3.4%)
Thr (T)11 (6.3%)
Trp (W)3 (1.7%)
Tyr (Y)6 (3.4%)
Val (V)18 (10.3%)
Amino acid counting
Total number:175
Positively charged (Arg+Lys):14 (8.0%)
Negatively charged (Asp+Glu):20 (11.4%)
Aromatic (Phe+His+Try+Tyr):21 (12.0%)
Biochemical parameters
Atomic composition:C882H1377N229O254S4
Molecular mass [Da]:19381.3
Theoretical pI:5.10
Extinction coefficient at 280 nm [M-1 cm-1]:25440 / 25503 (all Cys red/ox)
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.74)good (0.76)acceptable (0.55)good (0.65)excellent (0.81)good (0.77)
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Analytical präparation

[...] Characterization

Figure 3:
Figure 3:



Structure of the Nano Luciferase

Figure 3:
--->


XylE

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
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)
1 MAGNKGVMRPGHVQLRVLDMSKALEHYVELLGLIEMDRDDQGRVYLKAWTEVDKFSLVLREADEPGMDFMGFKVVDEDALRQLERDLMAYGCAVEQLPAG
101 ELNSCGRRVRFQAPSGHHFELYADKEYTGKWGLNDVNPEAWPRDLKGMAAVRFDHALMYGDELPATYDLFTKVLGFYLAEQVLDENGTRVAQFLSLSTKA
201 HDVAFIHHPEKGRLHHVSFHLETWEDLLRAADLISMTDTSIDIGPTRHGLTHGKTIYFFDPSGNRNEVFCGGDYNYPDHKPVTWTTDQLGKAIFYHDRIL
301 NERFMTVLTTG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
None of the supported features appeared in the sequence
Amino acid composition:
Ala (A)22 (7.1%)
Arg (R)19 (6.1%)
Asn (N)9 (2.9%)
Asp (D)27 (8.7%)
Cys (C)3 (1.0%)
Gln (Q)8 (2.6%)
Glu (E)21 (6.8%)
Gly (G)26 (8.4%)
His (H)15 (4.8%)
Ile (I)8 (2.6%)
Leu (L)34 (10.9%)
Lys (K)14 (4.5%)
Met (M)11 (3.5%)
Phe (F)16 (5.1%)
Pro (P)12 (3.9%)
Ser (S)10 (3.2%)
Thr (T)18 (5.8%)
Trp (W)5 (1.6%)
Tyr (Y)12 (3.9%)
Val (V)21 (6.8%)
Amino acid counting
Total number:311
Positively charged (Arg+Lys):33 (10.6%)
Negatively charged (Asp+Glu):48 (15.4%)
Aromatic (Phe+His+Try+Tyr):48 (15.4%)
Biochemical parameters
Atomic composition:C1585H2418N434O465S14
Molecular mass [Da]:35442.1
Theoretical pI:5.41
Extinction coefficient at 280 nm [M-1 cm-1]:45380 / 45568 (all Cys red/ox)
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):good (0.73)good (0.70)acceptable (0.55)good (0.63)excellent (0.82)good (0.78)
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Analytical präparation

[...] Characterization




DDT-Dehydrochlorinase

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
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)
1 MDFYYLPGSAPCRAVQMTAAAVGVELNLKLTDLMKGEHMKPEFLKLNPQHCIPTLVDNGFALWESRAIQIYLAEKYGKDDKLYPKDPQKRAVVNQRLYFD
101 MGTLYQRFADYHYPQIFAKQPANPENEKKMKDAVGFLNTFLEGQEYAAGNDLTIADLSLAATIATYEVAGFDFAPYPNVAAWFARCKANAPGYALNQAGA
201 DEFKAKFLS*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
None of the supported features appeared in the sequence
Amino acid composition:
Ala (A)30 (14.4%)
Arg (R)6 (2.9%)
Asn (N)11 (5.3%)
Asp (D)13 (6.2%)
Cys (C)3 (1.4%)
Gln (Q)10 (4.8%)
Glu (E)11 (5.3%)
Gly (G)12 (5.7%)
His (H)3 (1.4%)
Ile (I)6 (2.9%)
Leu (L)20 (9.6%)
Lys (K)16 (7.7%)
Met (M)6 (2.9%)
Phe (F)13 (6.2%)
Pro (P)13 (6.2%)
Ser (S)4 (1.9%)
Thr (T)8 (3.8%)
Trp (W)2 (1.0%)
Tyr (Y)13 (6.2%)
Val (V)9 (4.3%)
Amino acid counting
Total number:209
Positively charged (Arg+Lys):22 (10.5%)
Negatively charged (Asp+Glu):24 (11.5%)
Aromatic (Phe+His+Try+Tyr):31 (14.8%)
Biochemical parameters
Atomic composition:C1070H1618N272O304S9
Molecular mass [Da]:23444.8
Theoretical pI:5.94
Extinction coefficient at 280 nm [M-1 cm-1]:30370 / 30558 (all Cys red/ox)
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):excellent (0.87)excellent (0.85)good (0.62)good (0.71)excellent (0.80)good (0.69)
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Analytical präparation

[...] Characterization



PP1

Figure 3:
Figure 3:


SpyCatcher & SpyTag

[...] description [...] reaction [...] production

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
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)
1 MAGVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQ
101 GQVTVNGKATKGDAHITG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features)
None of the supported features appeared in the sequence
Amino acid composition:
Ala (A)10 (8.5%)
Arg (R)2 (1.7%)
Asn (N)2 (1.7%)
Asp (D)10 (8.5%)
Cys (C)0 (0.0%)
Gln (Q)5 (4.2%)
Glu (E)9 (7.6%)
Gly (G)14 (11.9%)
His (H)2 (1.7%)
Ile (I)6 (5.1%)
Leu (L)5 (4.2%)
Lys (K)8 (6.8%)
Met (M)3 (2.5%)
Phe (F)4 (3.4%)
Pro (P)2 (1.7%)
Ser (S)10 (8.5%)
Thr (T)14 (11.9%)
Trp (W)1 (0.8%)
Tyr (Y)4 (3.4%)
Val (V)7 (5.9%)
Amino acid counting
Total number:118
Positively charged (Arg+Lys):10 (8.5%)
Negatively charged (Asp+Glu):19 (16.1%)
Aromatic (Phe+His+Try+Tyr):11 (9.3%)
Biochemical parameters
Atomic composition:C543H848N144O192S3
Molecular mass [Da]:12561.7
Theoretical pI:4.49
Extinction coefficient at 280 nm [M-1 cm-1]:11460 / 11460 (all Cys red/ox)
Codon usage
Organism:E. coliB. subtilisS. cerevisiaeA. thalianaP. patensMammals
Codon quality (CAI):excellent (0.83)good (0.76)good (0.78)good (0.80)good (0.78)good (0.67)
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Analytical präparation

[...] Characterization


References:

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984

  1. 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.
  2. http://udel.edu/~gshriver/pdf/Pimenteletal1997.pdf Pmentel et al., 1997 Pimentel, D., Wilson, C., McCullum, C., Huang, R., Dwen, P., Flack, J. Tran, Q., Saltman, T., Cliff, T. (1997). Economic and environmental benefits of biodiversity. BioScience, Vol. 47, No. 11., pp. 747-757.




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