Difference between revisions of "Part:BBa K3075003"
 
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K3075001 short</partinfo>
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<partinfo>BBa_K3075003 short</partinfo>
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<partinfo>BBa_K3075003 SequenceAndFeatures</partinfo>
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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.  
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LXYL-p1-2-SpyT-His consists of the enzyme Beta-D-xylosidase fused to a C-terminal short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The additional SpyTag enables the enzyme to be attached to the Assemblase scaffold designed by the 2018 UNSW iGEM team via a Spy-Catcher-Tag conjugation system.  
  
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[[File:Part-BBa_K3075001-Introduction.png]]
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[[File:Lxyl_re.png]]
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'''Figure 1:''' A visualisation of the attachment of LXYL-p1-2-SpyT-His (green) fusion protein to the Assemblase scaffold (red) via the Spy-Catcher-Tag system. Graphic produced by Linda Chen 2019.
  
 
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)
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In Lentinula edodes, Beta-D-xylosidase (LXYL) catalyses the hydrolysis of O-glycosyl bonds. The native function of LXYL was to catalyse the reaction of 7-beta-xylosyl-10-deacetylbaccatin III (XDB) to 10-deacetylbaccatin III (DB). The LXYL-p1-2 mutant has been optimised to release the β-xylosyl group of 7-β-xylosyltaxanes (3). Specifically, the beta-hydrolase activity has been exploited to catalyse the removal of the xylose group from 7-beta-xylosyl-10-deacetyltaxol (XDT) to produce the intermediate 10-deacetyltaxol (DT) (3) (Figure 2). Recombinant LXYL-P1-2 has a sequence of 803 amino acid residues with a molecular mass of 85,975 Da.  
  
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=== Characterisation ===
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[[File:Lxyl_reaction2.png]]
  
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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 ??).  
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'''Figure 2:''' 7-beta-xylosidase catalyses the hydrolysis of 7-beta-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT). Figure obtained from Ping Zhu (2017).  
  
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Image
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=== Characterisation ===
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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.
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Figure 3. DBAT-SnoopT-His sequence chromatogram.
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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.
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Image
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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.
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'''Purification'''
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Following the confirmation of protein expression indicated by bug buster gels, attempts were made to purify DBAT.
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Image
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Figure ?. SDS-PAGE of AKTA purification fractions of DBAT His-tagged protein
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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.
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pET19b vector provided by Dr Dominic Glover 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 excess of insert. Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42°C and plated on ampicillin supplemented agar plates for selection. This resulted in seven (LXYL) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of LXYL transformants were screened by colony PCR, where LXYL colonies revealed bands of an estimated size 1000 bp, which is below the expected size of 2528 bp, showing the colonies did not contain the desired LXYL gene (Figure omitted).
  
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Image
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Assembly of LXYL was re-attempted, by increasing the insert to vector ratio to 5:1 and screening more colonies by colony PCR. Attempts did not succeed.
  
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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.
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As LXYL is a larger gene fragment (2.5kb), ligation into pET19b (5kb) would require a 2X excess of LXYL, instead of the 3X excess. However, excessive amounts of insert would unlikely to be the cause for non-assembly. It may be of benefit to increase the Gibson assembly incubation time to ensure there is sufficient time for the complementary overhangs to properly anneal between the insert and vector.
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
 
  
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=== References ===
  
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<!-- Uncomment this to enable Functional Parameter display
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#Lxyl-p1-2 - Beta-D-xylosidase/beta-D-glucosidase - Lentinula edodes (Shiitake mushroom) - Lxyl-p1-2 gene & protein [Internet]. Uniprot.org. 2019 [cited 22 October 2019]. Available from: https://www.uniprot.org/uniprot/G8GLP2
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===Functional Parameters===
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#Li B, Wang H, Gong T, Chen J, Chen T, Yang J et al. Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production. 2019.
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<partinfo>BBa_K3075001 parameters</partinfo>
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#Cheng H, Zhao R, Chen T, Yu W, Wang F, Cheng K et al. Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues. Molecular & Cellular Proteomics. 2013;12(8):2236-2248.
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<!-- -->
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#Chen J, Liang X, Wang F, Wen Y, Chen T, Liu W et al. Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast. Acta Pharmaceutica Sinica B. 2019;9(3):626-638.

Latest revision as of 02:24, 22 October 2019

LXYL-P1-2- SpyT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1062
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
    Illegal NgoMIV site found at 168
  • 1000
    COMPATIBLE WITH RFC[1000]


Introduction

LXYL-p1-2-SpyT-His consists of the enzyme Beta-D-xylosidase fused to a C-terminal short polypeptide tag (Spytag) and a Hexahistidine Tag (6xHis-tag), separated by interconnecting GSG linkage sequences. The additional SpyTag enables the enzyme to be attached to the Assemblase scaffold designed by the 2018 UNSW iGEM team via a Spy-Catcher-Tag conjugation system.

Lxyl re.png

Figure 1: A visualisation of the attachment of LXYL-p1-2-SpyT-His (green) fusion protein to the Assemblase scaffold (red) via the Spy-Catcher-Tag system. Graphic produced by Linda Chen 2019.

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

In Lentinula edodes, Beta-D-xylosidase (LXYL) catalyses the hydrolysis of O-glycosyl bonds. The native function of LXYL was to catalyse the reaction of 7-beta-xylosyl-10-deacetylbaccatin III (XDB) to 10-deacetylbaccatin III (DB). The LXYL-p1-2 mutant has been optimised to release the β-xylosyl group of 7-β-xylosyltaxanes (3). Specifically, the beta-hydrolase activity has been exploited to catalyse the removal of the xylose group from 7-beta-xylosyl-10-deacetyltaxol (XDT) to produce the intermediate 10-deacetyltaxol (DT) (3) (Figure 2). Recombinant LXYL-P1-2 has a sequence of 803 amino acid residues with a molecular mass of 85,975 Da.

Lxyl reaction2.png

Figure 2: 7-beta-xylosidase catalyses the hydrolysis of 7-beta-xylosyl-10-deacetyltaxol (XDT) to 10-deacetyltaxol (DT). Figure obtained from Ping Zhu (2017).

Characterisation

pET19b vector provided by Dr Dominic Glover 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 excess of insert. Gibson products were transformed into high efficiency T7 Express E. coli by heat shocking at 42°C and plated on ampicillin supplemented agar plates for selection. This resulted in seven (LXYL) transformant colonies, compared to zero colonies on the linear pET19b transformant negative control. Three colonies of LXYL transformants were screened by colony PCR, where LXYL colonies revealed bands of an estimated size 1000 bp, which is below the expected size of 2528 bp, showing the colonies did not contain the desired LXYL gene (Figure omitted).

Assembly of LXYL was re-attempted, by increasing the insert to vector ratio to 5:1 and screening more colonies by colony PCR. Attempts did not succeed.

As LXYL is a larger gene fragment (2.5kb), ligation into pET19b (5kb) would require a 2X excess of LXYL, instead of the 3X excess. However, excessive amounts of insert would unlikely to be the cause for non-assembly. It may be of benefit to increase the Gibson assembly incubation time to ensure there is sufficient time for the complementary overhangs to properly anneal between the insert and vector.


References

  1. Lxyl-p1-2 - Beta-D-xylosidase/beta-D-glucosidase - Lentinula edodes (Shiitake mushroom) - Lxyl-p1-2 gene & protein [Internet]. Uniprot.org. 2019 [cited 22 October 2019]. Available from: https://www.uniprot.org/uniprot/G8GLP2
  2. Li B, Wang H, Gong T, Chen J, Chen T, Yang J et al. Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production. 2019.
  3. Cheng H, Zhao R, Chen T, Yu W, Wang F, Cheng K et al. Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues. Molecular & Cellular Proteomics. 2013;12(8):2236-2248.
  4. Chen J, Liang X, Wang F, Wen Y, Chen T, Liu W et al. Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast. Acta Pharmaceutica Sinica B. 2019;9(3):626-638.