Difference between revisions of "Part:BBa K3075000"
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=== Introduction ===
 
=== Introduction ===
  
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DBAT-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.
+
PAM-Snooptag-His consists of the enzyme Phenylalanine aminomutase (PAM) fused at the C-terminus to a short polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of PAM originating from ''Taxus wallichiana'' var. chinensis was utilised. (1)
  
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[[File:Part-BBa_K3075001-Introduction.png]]
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[[File:PAM_Assemblase_pathway.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.
 
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.
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=== Usage and Biology ===
 
=== Usage and Biology ===
  
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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)
+
Phenylalanine aminomutase catalyses the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine. (2) Naturally, this enzyme is involved in the synthesis of trans-cinnamate from L-phenylalanine, an important part of Phenylpropanoid metabolism. It is also known to catalyse the initial step of N-benzoyl phenylisoserinoyl biosynthesis, which functions as the side chain of the anticancer drug Paclitaxel. (1) Recombinant phenylalanine aminomutase has a sequence of 698 amino acid residues with a molecular mass of 76,530 Daltons and maintains stability at a pH of 9-11 and temperature of 60-70C.
 +
 
 +
[[File:T--UNSW_Australia--Pathway1.png/800px-T--UNSW_Australia--Pathway1.png‎]]
  
 
=== Characterisation ===
 
=== Characterisation ===
  
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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 was 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 molar 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 two (PAM) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control.
 +
 
 +
Two colonies of PAM transformants were screened by colony PCR, where the PAM-2 colony exhibited a single band close to the estimated length of 2.18 kb (Figure 5). PAM-2 colony was grown overnight in a 5 mL culture and plasmid DNA was extracted via miniprep. Samples were submitted for sequence confirmation by Sanger sequencing.
 +
 
 +
[[File:T--UNSW_Australia--OamPCRgel.png‎]]
 +
 
 +
Figure 5. Colony PCR gel image of recombinant PAM-pET19b plasmid. PAM gene was amplified by colony PCR at Tm = 67.6°C and extension time 50 seconds, else as per protocol <link>. 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 ~2.2 kb.
 +
 
 +
Sequence alignment reveal sequence homology of the PAM-2 colony with our designed gene construct. Thus, PAM has been successfully cloned into pET19b backbone. However, the gene contains five base inaccuracies. This results in the following amino acid changes: silent mutations at amino acid residue 123 (P) and 713 (Stop), and a missense mutation R106C (Table 1). These detected base changes could be a result of a sequencing error, errors introduced during PCR or errors in the original purchased gene fragment.
 +
 
 +
Table 1: Base inconsistencies between Sanger sequencing results of PAM-2 and the desired PAM-SnoopT sequence.
  
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Image
+
[[File:PAM_Sequencing_Table.PNG]]
  
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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.
+
To completely understand the effect of the R106C change, further research into the location of this amino acid within the protein by modelling or otherwise could be completed. This would help determine whether the amino acid change would have interfered in the protein folding, or substrate binding of PAM.
  
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Figure 3. DBAT-SnoopT-His sequence chromatogram.
+
=== Protein Expression and Purification ===
  
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'''Protein expression assay'''
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'''Protein Expression assay'''
  
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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.
+
Cells containing a plasmid with the PAM insert (as confirmed by colony PCR and sequencing) were grown up with a sample of this used to perform a protein expression assay. Bug buster was then used to separate the soluble and insoluble proteins.  
  
−
Image
+
[[File:T--UNSW_Australia--7-PAM-bug-buster.jpeg/384px-T--UNSW_Australia--7-PAM-bug-buster.jpeg]]
  
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Figure ?. Protein expression assay using bug buster to determine expression of 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) as soluble and insoluble form.
+
Figure 7. Protein expression assay using bug buster to determine expression of Phenylalanine aminomutase (PAM) as soluble and insoluble form.
  
 
'''Purification'''
 
'''Purification'''
  
−
Following the confirmation of protein expression indicated by bug buster gels, attempts were made to purify DBAT.
+
Following the confirmation of protein expression using bug buster gels in Figure 7, attempts were made to purify PAM. The eluted fractions were concentrated 20-fold using an Amicon Ultra-0.5 mL Centrifugal Filters to increase the visibility of any soluble PAM present.
  
−
Image
+
[[File:T--UNSW_Australia--8-PAM-purification.jpeg/800px-T--UNSW_Australia--8-PAM-purification.jpeg]]
  
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Figure ?. SDS-PAGE of AKTA purification fractions of DBAT His-tagged protein
+
Figure 8. SDS-PAGE of AKTA purification fractions (F3 to F6) for PAM His-tagged protein
  
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'''Liquid Chromatography with tandem mass spectrometry'''
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'''Liquid Chromatography with tandem Mass Spectrometry'''
  
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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.
+
Soluble protein bands (concentrated fractions 4-6) and total protein lysate bands were excised from the gel of purified fractions seen in Figure 8. These bands were the same predicted molecular weight as PAM. The bands were sent for analysis by Liquid Chromatography with tandem Mass Spectrometry (LC-MS-MS). This was performed to determine the identity of the protein bands by mapping peptides detected by LC-MS-MS onto the sequence of PAM obtained from sequencing data of the cloned insert.
  
−
Image
+
[[File:T--UNSW_Australia--9-PAM-Mass-Spec.jpeg/800px-T--UNSW_Australia--9-PAM-Mass-Spec.jpeg]]
  
−
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.
+
Figure 9.  LCMSMS analysis of suspected PAM protein bands excised form Figure 8 protein gel. A: Total protein lysate sample. B: Soluble protein sample taken from concentrated fractions 4-6.
  
 
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Revision as of 06:55, 21 October 2019

PAM-SnoopT-His

Introduction

PAM-Snooptag-His consists of the enzyme Phenylalanine aminomutase (PAM) fused at the C-terminus to a short polypeptide tag (Snooptag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The sequence of PAM originating from Taxus wallichiana var. chinensis was utilised. (1)

PAM Assemblase pathway.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

Phenylalanine aminomutase catalyses the conversion of 2S-α-phenylalanine to 3R-β-phenylalanine. (2) Naturally, this enzyme is involved in the synthesis of trans-cinnamate from L-phenylalanine, an important part of Phenylpropanoid metabolism. It is also known to catalyse the initial step of N-benzoyl phenylisoserinoyl biosynthesis, which functions as the side chain of the anticancer drug Paclitaxel. (1) Recombinant phenylalanine aminomutase has a sequence of 698 amino acid residues with a molecular mass of 76,530 Daltons and maintains stability at a pH of 9-11 and temperature of 60-70C.

File:T--UNSW Australia--Pathway1.png/800px-T--UNSW Australia--Pathway1.png

Characterisation

pET19b vector was provided by Dr Dominic Glover and was 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 molar 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 two (PAM) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control.

Two colonies of PAM transformants were screened by colony PCR, where the PAM-2 colony exhibited a single band close to the estimated length of 2.18 kb (Figure 5). PAM-2 colony was grown overnight in a 5 mL culture and plasmid DNA was extracted via miniprep. Samples were submitted for sequence confirmation by Sanger sequencing.

File:T--UNSW Australia--OamPCRgel.png

Figure 5. Colony PCR gel image of recombinant PAM-pET19b plasmid. PAM gene was amplified by colony PCR at Tm = 67.6°C and extension time 50 seconds, else as per protocol <link>. 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 ~2.2 kb.

Sequence alignment reveal sequence homology of the PAM-2 colony with our designed gene construct. Thus, PAM has been successfully cloned into pET19b backbone. However, the gene contains five base inaccuracies. This results in the following amino acid changes: silent mutations at amino acid residue 123 (P) and 713 (Stop), and a missense mutation R106C (Table 1). These detected base changes could be a result of a sequencing error, errors introduced during PCR or errors in the original purchased gene fragment.

Table 1: Base inconsistencies between Sanger sequencing results of PAM-2 and the desired PAM-SnoopT sequence.

PAM Sequencing Table.PNG

To completely understand the effect of the R106C change, further research into the location of this amino acid within the protein by modelling or otherwise could be completed. This would help determine whether the amino acid change would have interfered in the protein folding, or substrate binding of PAM.

Protein Expression and Purification

Protein Expression assay

Cells containing a plasmid with the PAM insert (as confirmed by colony PCR and sequencing) were grown up with a sample of this used to perform a protein expression assay. Bug buster was then used to separate the soluble and insoluble proteins.

File:T--UNSW Australia--7-PAM-bug-buster.jpeg/384px-T--UNSW Australia--7-PAM-bug-buster.jpeg

Figure 7. Protein expression assay using bug buster to determine expression of Phenylalanine aminomutase (PAM) as soluble and insoluble form.

Purification

Following the confirmation of protein expression using bug buster gels in Figure 7, attempts were made to purify PAM. The eluted fractions were concentrated 20-fold using an Amicon Ultra-0.5 mL Centrifugal Filters to increase the visibility of any soluble PAM present.

File:T--UNSW Australia--8-PAM-purification.jpeg/800px-T--UNSW Australia--8-PAM-purification.jpeg

Figure 8. SDS-PAGE of AKTA purification fractions (F3 to F6) for PAM His-tagged protein

Liquid Chromatography with tandem Mass Spectrometry

Soluble protein bands (concentrated fractions 4-6) and total protein lysate bands were excised from the gel of purified fractions seen in Figure 8. These bands were the same predicted molecular weight as PAM. The bands were sent for analysis by Liquid Chromatography with tandem Mass Spectrometry (LC-MS-MS). This was performed to determine the identity of the protein bands by mapping peptides detected by LC-MS-MS onto the sequence of PAM obtained from sequencing data of the cloned insert.

File:T--UNSW Australia--9-PAM-Mass-Spec.jpeg/800px-T--UNSW Australia--9-PAM-Mass-Spec.jpeg

Figure 9. LCMSMS analysis of suspected PAM protein bands excised form Figure 8 protein gel. A: Total protein lysate sample. B: Soluble protein sample taken from concentrated fractions 4-6.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 498
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 498
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 498
    Illegal BamHI site found at 2131
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 498
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 498
    Illegal NgoMIV site found at 393
  • 1000
    COMPATIBLE WITH RFC[1000]