Difference between revisions of "Part:BBa K3075001"
 
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<partinfo>BBa_K3075001 short</partinfo>
 
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<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
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=== Introduction ===
 
=== Introduction ===
  
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DDBAT-Snooptag-His consists of the enzyme 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) fused to a short C-terminal polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of DBAT which was used, originated from ''Taxus cuspidata'' (Japanese yew), with a double mutation of G38R/F301V (2). The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.  
+
DBAT<sup>G38R/F301V</sup>-SnoopT-His consists of the enzyme 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) fused to a short C-terminal polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of DBAT which was used, originated from Taxus cuspidata (Japanese yew), with a double mutation of G38R/F301V (2). The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.  
  
−
Image
+
[[File:Part-BBa_K3075001-Introduction.png]]
  
−
The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.
+
'''Figure 1:'''
  
 +
The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.
  
 
=== Usage and Biology ===
 
=== Usage and Biology ===
  
−
This protein naturally participates in the synthesis of baccatin III, where it catalyses the final acetylation of 10-deacetylbaccatin III. Baccatin III synthesis is a subpathway of paclitaxel biosynthesis, which is itself part of Alkaloid biosynthesis. The mutant however, has been designed to catalyse the acetylation of 10-deacetyltaxol (DT) with a catalytic efficiency approximately six times higher than that of the wild-type. (2) The recombinant mutant enzyme has a length of 440 amino acid residues, a molecular weight of 49,052 Da and an optimum pH of 7.5. (3)
+
This protein naturally participates in the synthesis of baccatin III from 10-deacetyl-2-debenzoylbaccatin III, where it catalyses the final acetylation. Baccatin III synthesis is a subpathway of paclitaxel biosynthesis, which is part of Alkaloid biosynthesis. The mutant however, has been designed to catalyse the acetylation of 10-deacetyltaxol (DT) with a catalytic efficiency approximately six times higher than that of the wild-type. (2) The recombinant mutant enzyme has a length of 440 amino acid residues, a molecular weight of 49,052 Da and an optimum pH of 7.5. (3)
 +
 
 +
[[File:DBAT_LXYL_diagram.png]]
 +
 
 +
'''Figure 2:'''
  
 
=== Characterisation ===
 
=== Characterisation ===
  
−
The gBlock was assembled into the pET19b expression vector at the multiple cloning site via gibson assembly with a 3-fold excess of insert to vector <link to protocols>. Gibson products were transformed into high efficiency T7 Express E. coli (NEB) by heat shocking at 42°C and cells were plated on ampicillin supplemented agar plates for selection. Transformants were screened for recombinant plasmids by colony PCR (figure ??). Colonies resulting in amplicons with an observed molecular weight of approximately 1.5 kb were grown overnight in a 5 mL culture and plasmid DNA was extracted by miniprep and submitted for sequence confirmation via Sanger sequencing (Figure ??).  
+
pET19b vector was provided by Dr Dominic Glover and linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA technologies (IDT). The gene constructs were assembled into the pET19b expression vector at the multiple cloning site via Gibson assembly with a 3-fold excess of insert.
  
−
Image
+
Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42C and plated on ampicillin supplemented agar plates for selection. This resulted in nine (DBAT) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of DBAT were screened by colony PCR, where DBAT-1 obtained a single band of estimated molecular weight ______ (approx. 1.4kb)[MT1]  (Figure 3).
  
−
Figure 2. Recombinant DBAT-SnoopT-His gene amplified by colony PCR at annealing temperature 67.6°C and extension time 43 seconds, else as per protocol. 10 uL of PCR product was run on a 1% agarose gel at 100 V for 1 hour using 5 uL of 2-log DNA ladder (NEB) as a standard (Lane 1). Single band at ~1.5kb.
+
[[File:DBAT_PCR_gel.png]]
  
−
Figure 3. DBAT-SnoopT-His sequence chromatogram.
+
'''Figure 3:'''
  
−
'''Protein expression assay'''
+
DBAT-1 colony was grown overnight in a 5mL culture and plasmid DNA was extracted by miniprep. Samples were submitted for sequence confirmation by Sanger sequencing, obtaining the sequence utilising forward and reverse primers. Sequence alignment revealed 100% sequence homology of the DBAT-1 colony. The DBAT<sup>G38R/F301V</sup>-SnoopT-His gene fragment was aligned from base 23 to 1195 (in the D1-F sequence alignment file) and ____ to _____ [MT3] (in the D1-R sequence alignment file). This confirms that DBAT<sup>G38R/F301V</sup>-SnoopT-His has been successfully assembled and transformed within DBAT-1 colony and was stored in glycerol stocks and prepared for protein expression and purification.
  
−
Cells containing a plasmid with the DBAT insert were grown up and a sample of this was used to perform a protein expression assay. Bug buster was used to separate soluble and insoluble proteins. LXYL was not successfully cloned thus could not be expressed.
+
=== Protein Expression and Purification ===
  
−
Image
+
==== Protein expression assay ====
  
−
Figure ?. Protein expression assay using bug buster to determine expression of 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) as soluble and insoluble form.
+
Cells containing a plasmid with the DBAT insert were grown up with a sample of this being used to perform a protein expression assay. Additionally, Bug buster was used to separate soluble and insoluble proteins.  
  
−
'''Purification'''
+
[[File:DBAT_Bug_Buster.png]]
  
−
Following the confirmation of protein expression indicated by bug buster gels, attempts were made to purify DBAT.
+
'''Figure 4:''' Protein expression assay using bug buster to determine expression of 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) as soluble and insoluble form.
  
−
Image
+
==== Purification ====
  
−
Figure ?. SDS-PAGE of AKTA purification fractions of DBAT His-tagged protein
+
Following the confirmation of protein expression using bug buster gels, attempts were made to purify DBAT<sup>G38R/F301V</sup>-SnoopT-His.
  
−
'''Liquid Chromatography with tandem mass spectrometry'''
+
[[File:DBAT_SDS_Page.png]]
  
−
Soluble protein bands (fractions 4-7) as well as a total protein lysate band at the same predicted molecular weight as DBAR were excised from the gel of purified fractions in Figure? And sent for analysis by Liquid Chromatography with tandem mass spectrometry (LCMSMS). This was performed to determine the identity of the protein bands by mapping peptides detected by LCMSMS onto the sequence of DBAT obtained from sequencing data of the cloned insert.
+
'''Figure 5:''' SDS-PAGE of AKTA purification fractions of DBAT His-tagged protein
  
−
Image
+
==== Liquid Chromatography with tandem mass spectrometry ====
  
−
Figure ?.  Liquid Chromatography with tandem mass spectrometry analysis of suspected DBAT protein bands excises form Figure ? protein gel. A: Total protein lysate sample. B: soluble protein sample taken from fractions 4-7.
+
Soluble protein bands (fractions 4-7) as well as, a total protein lysate band at the same predicted molecular weight as DBAT were excised from the Figure 5 gel and sent for analysis. Trypsin digest followed by liquid chromatography- tandem mass spectrometry (LC-MS/MS) confirmed the identity of the protein bands as DBAT, the peptides of which are shown in figure 6.
 +
 
 +
[[File:DBAT_Mass_Spec.png]]
 +
 
 +
'''Figure 6:''' The Liquid Chromatography with Tandem Mass Spectrometry analysis of suspected DBAT protein bands excised form Figure ? protein gel. '''A:''' Total protein lysate sample. '''B:''' Soluble protein sample taken from fractions 4-7.
 +
 
 +
=== Validation ===
 +
 
 +
==== Ellman’s Reagent Assay ====
 +
 
 +
A standard curve measuring absorbance vs differing concentrations of CoA-SH was constructed (Figure 7) with a computer generated linear trendline.
 +
 
 +
[[File:Ellman's_Standard.png]]
 +
 
 +
'''Figure 7:''' Standard curve of TNB absorbance measured at differing CoA-SH concentrations. Included is the computer generated linear trendline with Cartesian equation and R value.
 +
 
 +
A discontinuous assay was conducted in triplicate at 15-minute interval time points for a total of 90 minutes. Mean sample absorbance – Mean blank absorbance was calculated for each time point. Excel was then used to create a graph containing a computer-generated linear trendline (figure 8)
 +
 
 +
[[File:DBAT_Ellman's.png]]
 +
 
 +
'''Figure 8:''' Endpoint assay of change in TNB absorbance over 90 minutes, measured every 15 minutes. Included is the computer generated linear trendline with Cartesian equation and R value
 +
 
 +
The amount of TNB produced in 1 minute was calculated by c= ∆A/(εl) where c is the concentration change of TNB (M), ∆A is the change in absorbance in 1 minute, molar extinction coefficient ε= 14 150 M-1cm-1, and pathlength l= 1 cm. (2) The result was then divided by 60 to get the change in TNB concentration per second. (3)
 +
 
 +
The number of moles of TNB produced per second was calculated by n= cV where c is the concentration change calculated from the step above, and V = 350 x 10-6 L.
 +
 
 +
The rate of catalysis was calculated by (moles of TNB produced per second)/(moles of enzyme) as TNB is formed in a 1:1 molar ratio with CoA-SH produced. This gave us an estimated Kcat of 0.0056 (1/s)
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
 
  
 +
=== References ===
  
−
<!-- Uncomment this to enable Functional Parameter display
+
#Walker KD, Klettke K, Akiyama T, Croteau R. Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis. Journal of Biological Chemistry. 2004 Dec 24;279(52):53947-54.
−
===Functional Parameters===
+
#Woestenenk EA, Hammarström M, van den Berg S, Härd T, Berglund H. His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors. Journal of structural and functional genomics. 2004 Sep 1;5(3):217-29.
−
<partinfo>BBa_K3075001 parameters</partinfo>
+
#WEEK #3: ENZYME KINETICS: CHARACTERIZATION OF THE ENZYME ALKALINE PHOSPHATASE [Internet]. [cited 19 October 2019]. Available from: https://acad.carleton.edu/curricular/BIOL/classes/bio126/Documents/Lab_3.pdf?fbclid=IwAR1REQ1CFrBmPqrPmGXsg7E8N6s-bjxedr_VyICuEBsCa7hUEsTikM7i5aQ
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<!-- -->
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Latest revision as of 02:18, 22 October 2019


DBATG38R/F301V-SnoopT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 28
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

DBATG38R/F301V-SnoopT-His consists of the enzyme 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) fused to a short C-terminal polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of DBAT which was used, originated from Taxus cuspidata (Japanese yew), with a double mutation of G38R/F301V (2). The SnoopTag is a small polypeptide tag that spontaneously forms an isopeptide bond between reactive amino acid side chains to its corresponding SnoopCatcher (Brune, 2017). This system opens up a variety of applications, utilising the catcher-tag conjugation system for bioconjugation and synthetic assembly of the DBAT enzyme to SnoopCatcher containing proteins.

Part-BBa K3075001-Introduction.png

Figure 1:

The Hexahistidine tag is a common additive due to its high affinity for metal ions used in the purification technique of immobilized metal affinity chromatography (IMAC). Ni2+ ions were used for his-tag purification due to its high yield.

Usage and Biology

This protein naturally participates in the synthesis of baccatin III from 10-deacetyl-2-debenzoylbaccatin III, where it catalyses the final acetylation. Baccatin III synthesis is a subpathway of paclitaxel biosynthesis, which is part of Alkaloid biosynthesis. The mutant however, has been designed to catalyse the acetylation of 10-deacetyltaxol (DT) with a catalytic efficiency approximately six times higher than that of the wild-type. (2) The recombinant mutant enzyme has a length of 440 amino acid residues, a molecular weight of 49,052 Da and an optimum pH of 7.5. (3)

DBAT LXYL diagram.png

Figure 2:

Characterisation

pET19b vector was provided by Dr Dominic Glover and linearised by PCR amplification. Linear gene fragments were purchased from Integrated DNA technologies (IDT). The gene constructs were assembled into the pET19b expression vector at the multiple cloning site via Gibson assembly with a 3-fold excess of insert.

Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42C and plated on ampicillin supplemented agar plates for selection. This resulted in nine (DBAT) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of DBAT were screened by colony PCR, where DBAT-1 obtained a single band of estimated molecular weight ______ (approx. 1.4kb)[MT1] (Figure 3).

DBAT PCR gel.png

Figure 3:

DBAT-1 colony was grown overnight in a 5mL culture and plasmid DNA was extracted by miniprep. Samples were submitted for sequence confirmation by Sanger sequencing, obtaining the sequence utilising forward and reverse primers. Sequence alignment revealed 100% sequence homology of the DBAT-1 colony. The DBATG38R/F301V-SnoopT-His gene fragment was aligned from base 23 to 1195 (in the D1-F sequence alignment file) and ____ to _____ [MT3] (in the D1-R sequence alignment file). This confirms that DBATG38R/F301V-SnoopT-His has been successfully assembled and transformed within DBAT-1 colony and was stored in glycerol stocks and prepared for protein expression and purification.

Protein Expression and Purification

Protein expression assay

Cells containing a plasmid with the DBAT insert were grown up with a sample of this being used to perform a protein expression assay. Additionally, Bug buster was used to separate soluble and insoluble proteins.

DBAT Bug Buster.png

Figure 4: Protein expression assay using bug buster to determine expression of 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) as soluble and insoluble form.

Purification

Following the confirmation of protein expression using bug buster gels, attempts were made to purify DBATG38R/F301V-SnoopT-His.

DBAT SDS Page.png

Figure 5: SDS-PAGE of AKTA purification fractions of DBAT His-tagged protein

Liquid Chromatography with tandem mass spectrometry

Soluble protein bands (fractions 4-7) as well as, a total protein lysate band at the same predicted molecular weight as DBAT were excised from the Figure 5 gel and sent for analysis. Trypsin digest followed by liquid chromatography- tandem mass spectrometry (LC-MS/MS) confirmed the identity of the protein bands as DBAT, the peptides of which are shown in figure 6.

DBAT Mass Spec.png

Figure 6: The Liquid Chromatography with Tandem Mass Spectrometry analysis of suspected DBAT protein bands excised form Figure ? protein gel. A: Total protein lysate sample. B: Soluble protein sample taken from fractions 4-7.

Validation

Ellman’s Reagent Assay

A standard curve measuring absorbance vs differing concentrations of CoA-SH was constructed (Figure 7) with a computer generated linear trendline.

Ellman's Standard.png

Figure 7: Standard curve of TNB absorbance measured at differing CoA-SH concentrations. Included is the computer generated linear trendline with Cartesian equation and R value.

A discontinuous assay was conducted in triplicate at 15-minute interval time points for a total of 90 minutes. Mean sample absorbance – Mean blank absorbance was calculated for each time point. Excel was then used to create a graph containing a computer-generated linear trendline (figure 8)

DBAT Ellman's.png

Figure 8: Endpoint assay of change in TNB absorbance over 90 minutes, measured every 15 minutes. Included is the computer generated linear trendline with Cartesian equation and R value

The amount of TNB produced in 1 minute was calculated by c= ∆A/(εl) where c is the concentration change of TNB (M), ∆A is the change in absorbance in 1 minute, molar extinction coefficient ε= 14 150 M-1cm-1, and pathlength l= 1 cm. (2) The result was then divided by 60 to get the change in TNB concentration per second. (3)

The number of moles of TNB produced per second was calculated by n= cV where c is the concentration change calculated from the step above, and V = 350 x 10-6 L.

The rate of catalysis was calculated by (moles of TNB produced per second)/(moles of enzyme) as TNB is formed in a 1:1 molar ratio with CoA-SH produced. This gave us an estimated Kcat of 0.0056 (1/s)


References

  1. Walker KD, Klettke K, Akiyama T, Croteau R. Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis. Journal of Biological Chemistry. 2004 Dec 24;279(52):53947-54.
  2. Woestenenk EA, Hammarström M, van den Berg S, Härd T, Berglund H. His tag effect on solubility of human proteins produced in Escherichia coli: a comparison between four expression vectors. Journal of structural and functional genomics. 2004 Sep 1;5(3):217-29.
  3. WEEK #3: ENZYME KINETICS: CHARACTERIZATION OF THE ENZYME ALKALINE PHOSPHATASE [Internet]. [cited 19 October 2019]. Available from: https://acad.carleton.edu/curricular/BIOL/classes/bio126/Documents/Lab_3.pdf?fbclid=IwAR1REQ1CFrBmPqrPmGXsg7E8N6s-bjxedr_VyICuEBsCa7hUEsTikM7i5aQ