Difference between revisions of "Part:BBa K2688003"
Line 15: Line 15:
 
<partinfo>BBa_K2688003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2688003 SequenceAndFeatures</partinfo>
  
 +
 +
 +
== Expression vector design ==
 +
[[File:T--GO_Paris-Saclay--folc_cpg2_pSB1C3.png|300px|thumb|right| Figure 2: Genetic organization of the folC-cpg2 encoding vector (BBa_K2688009)]]
 +
We engineered a two-component pathway for efficient MTX degradation with ''folC'' and ''cpg2'' under inducible ''ara'' (AraO2 BBa_R0081 + AraC BBa_R0080) promoter and a phage lambda pL promoter with ''lacO'' sites (BBa_R0011) ''lac'', respectively. The expression of their respective repressors (AraC and LacI) by ''E. coli'' K12 strains allows the inducible expression in presence of arabinose (0.2 %) and IPTG (0,5 mM).
 +
 +
The CPG2 protein encoding sequence from ''Pseudomonas'' strain RS-16 was modified in two ways:
 +
 +
*  The periplasmic localisation signal was removed
 +
** EMBOSS software detects hydrophobic signal at amino acid 13->25 or 12->24
 +
** Decision was taken to remove AA 1 to 22 to achieve protein sequence identity with the commercially available CPG2 (drug INN glucarpidase)
 +
* The sequence was codon-optimzed for an optimal expression in ''E. coli'' (%GC, illegal restriction site and codon bias)
 +
 +
The synthetic ''cpg2'' gene was cloned within pSB1C3, alone or associated the ''folC'' expression cassette ('''Figure 2''').
 +
 +
 +
== Characterization ==
 +
'''Summary of methods''': We aimed to evaluate the activity of our degradation pathway, by incubating a degrader strain containing ''cpg2'' encoding plasmid in LB medium containing presence of 512 µM MTX. The culture supernatant was harvested and filtered before assessing the residual MTX either by a bioassay (bacteria particularly sensitive to MTX) or HPLC.
 +
 +
'''Results summary''': This characterization was successful, with both the bioassay and HPLC reporting almost complete MTX degradation. After only 5 h of incubation with bacteria harbouring a ''cpg2'' encoding plasmid, the MTX was almost completely removed from the medium (HPLC analysis).
 +
 +
=== Materials and methods ===
 +
'''Strains and plasmids'''
 +
 +
Two categories of strains were used: degrader strains (‘chassis’ harbouring ''cpg2'' encoding plasmid), and indicative strains with high MTX sensitivity, as biosensor for the biological effects of MTX. All strains were derived from ''E. coli'' K12 BW25113 (''Δ(araD-araB)567'', ''ΔlacZ4787''(::rrnB-3), ''λ<sup>-</sup>'', ''rph-1'', ''Δ(rhaD-rhaB)568'', ''hsdR514'') parental strain and obtained from the Keio mutant collection<ref name="Baba2006" />.
 +
 +
For characterization purpose, two degrader strains were selected: WT and Δ''tolC'' (Keio mutant JW5503). The latter has an intrinsic growth defect, but its lower MIC (minimum inhibitory concentration) for MTX<ref name="Kopytek2000" /> made it a possibly better degrader chassis. We used ''ΔacrA'' mutant (Keio JW0452) as an indicative strain since it is clearly MTX-sensitive.
 +
 +
We analysed MXT biotransformation by WT and Δ''tolC'' strains harbouring pSB1C3-''cpg2'' (BBa_K2688003) or pSB1C3-''folC-cpg2'' (BBa_K2688009).  Strains containing pSB1C3-''tet'' (BBa_R0040)  or pSB1C3-''folC'' (BBa_K2688008) were used as controls.
 +
 +
'''MTX-biotransformation assays'''
 +
 +
The MTX degradation experiments were performed in LB medium containing 0.5 mM IPTG and 0.2% (m/v) arabinose supplemented with: i) 2% v/v DMSO (which is the solvent we used to prepare MTX stock solution) for the control condition, or ii) with512 µM MTX (kindly provided by Raphaël Labruère).
 +
 +
The degradation experiments were conducted in 15 mL Falcon tubes, with 1 mL of either control or MTX medium, inoculated with the strains at an initial OD of 0.2. The tubes were incubated for 5  to 20 h at 37°C in ambient air. Afterward, the cultures were centrifuged 15 min at 5400 ''g''. The supernatants were filtered (0.22 µM) to eliminate cells and obtain an evaluable supernatant filtrate.
 +
 +
To assess the quantity of MTX remaining, two assays were conducted: HPLC and MTX bioassay.
 +
 +
'''MTX-detection bioassays'''
 +
 +
The MTX bioassay was a viability experiment using the ΔacrA indicative strain cultured in 96-wells plate with 50 µL of filtrate and 50 µL Δ''acrA'' (OD 0.04 obtained from an overnight culture in LB).
 +
 +
This viability experiment was conducted in a 96-wells plate over 700 min in a Clariostar® plate reader (''BMG LABtech GmbH''), with orbital shaking, temperature control at 37°C and ambient air atmosphere, measuring OD<sub>600</sub> every 20 min. The transparent plates were sealed with an adhesive transparent top. Duplicate or triplicate were performed for each condition.
 +
 +
'''HPLC analysis'''
 +
 +
The HPLC analysis was performed using a reverse phase C18 column. Detection was assessed by a UV spectrophotometer at 303 nm (peak absorption 302-303 nm<ref name="Merck2007" />) with peak-triggered UV-vis spectrum (diode array). The eluting solvent was a gradient of acetonitrile and water (with 0.1% v/v HCOOH). This mode of operation is very similar to the assay and impurities protocols of reference pharmacopeia for MTX. <ref name="PhEur" /><ref name="USP-NF" />
  
  
Line 32: Line 79:
 
<ref name="Masterson2006">Masterson, L. A., Spanswick, V. J., Hartley, J. A., Begent, R. H., Howard, P. W., & Thurston, D. E. (2006). Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy. Bioorganic & Medicinal Chemistry Letters, 16(2), 252–256. https://doi.org/10.1016/J.BMCL.2005.10.017</ref>
 
<ref name="Masterson2006">Masterson, L. A., Spanswick, V. J., Hartley, J. A., Begent, R. H., Howard, P. W., & Thurston, D. E. (2006). Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy. Bioorganic & Medicinal Chemistry Letters, 16(2), 252–256. https://doi.org/10.1016/J.BMCL.2005.10.017</ref>
  
 +
 +
<ref name="Baba2006">Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., … Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Molecular Systems Biology, 2, 2006.0008. https://doi.org/10.1038/msb4100050</ref>
 +
 +
<ref name="Kopytek2000">Kopytek, S. J., Dyer, J. C. D., Knapp, G. S., & Hu, J. C. (2000). Resistance to methotrexate due to AcrAB-dependent export from Escherichia coli. Antimicrobial Agents and Chemotherapy, 44(11), 3210–3212. https://doi.org/10.1128/AAC.44.11.3210-3212.2000</ref>
 +
 +
<ref name="Merck2007">Merck. (2007). The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 14th. Journal of the American Chemical Society, 129(7), 2197–2197. https://doi.org/10.1021/ja069838y</ref>
 
</references>
 
</references>
 +
 +
<ref name="PhEur">Council of Europe. European Pharmacopoeia. 6th ed. Strasbourg: Council of
 +
Europe; 2014. Methotrexate; p. 2735.</ref>
 +
 +
<ref name="USP-NF">United States Pharmacopeia and National Formulary (USP 30-NF 25). Vol 2. Rockville, MD: United States Pharmacopeial Convention; 2007:1553-1554.</ref>

Revision as of 18:18, 12 October 2018

cpg2_tu

Executive summary : This BioBrick is a full translation unit encoding CPG2, an enzyme that converts the anticancer drug methotrexate (MTX) into less toxic compounds. When expressed in E. coli, we have shown that it can rapidly remove MTX from the culture medium. This is proven conclusively by HPLC, and a bioassay that measures residual toxicity.

The Pseudomonas carboxypeptidase G2 (CPG2 or ‘glucarpidase’) is an hydrolase that cleaves the (poly)glutamate tail off folates and analogs, leaving behind a pteroate ring system[1]). There is broad substrate specificity, including endogenous folates, and notably the chemotherapeutic drug MTX (MTX). Its metabolite DAMPA (2,4-diamino-N10-methylpteroic acid) (Figure 1) has little antifolate activity in vitro, and norelevant clinical effects compared to MTX[2].

This has led to its use as both an antidote in case of MTX poisoning[3], and as a general purpose platform for novel drug delivery methods, where CPG22 would activate polyglutamated, soluble prodrugs in situ[4].

Figure 1 : The MTX biotransformation by Pseudomonas CPG2


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 132
    Illegal NgoMIV site found at 820
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 919


Expression vector design

Figure 2: Genetic organization of the folC-cpg2 encoding vector (BBa_K2688009)

We engineered a two-component pathway for efficient MTX degradation with folC and cpg2 under inducible ara (AraO2 BBa_R0081 + AraC BBa_R0080) promoter and a phage lambda pL promoter with lacO sites (BBa_R0011) lac, respectively. The expression of their respective repressors (AraC and LacI) by E. coli K12 strains allows the inducible expression in presence of arabinose (0.2 %) and IPTG (0,5 mM).

The CPG2 protein encoding sequence from Pseudomonas strain RS-16 was modified in two ways:

  • The periplasmic localisation signal was removed
    • EMBOSS software detects hydrophobic signal at amino acid 13->25 or 12->24
    • Decision was taken to remove AA 1 to 22 to achieve protein sequence identity with the commercially available CPG2 (drug INN glucarpidase)
  • The sequence was codon-optimzed for an optimal expression in E. coli (%GC, illegal restriction site and codon bias)

The synthetic cpg2 gene was cloned within pSB1C3, alone or associated the folC expression cassette (Figure 2).


Characterization

Summary of methods: We aimed to evaluate the activity of our degradation pathway, by incubating a degrader strain containing cpg2 encoding plasmid in LB medium containing presence of 512 µM MTX. The culture supernatant was harvested and filtered before assessing the residual MTX either by a bioassay (bacteria particularly sensitive to MTX) or HPLC.

Results summary: This characterization was successful, with both the bioassay and HPLC reporting almost complete MTX degradation. After only 5 h of incubation with bacteria harbouring a cpg2 encoding plasmid, the MTX was almost completely removed from the medium (HPLC analysis).

Materials and methods

Strains and plasmids

Two categories of strains were used: degrader strains (‘chassis’ harbouring cpg2 encoding plasmid), and indicative strains with high MTX sensitivity, as biosensor for the biological effects of MTX. All strains were derived from E. coli K12 BW25113 (Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568, hsdR514) parental strain and obtained from the Keio mutant collection[5].

For characterization purpose, two degrader strains were selected: WT and ΔtolC (Keio mutant JW5503). The latter has an intrinsic growth defect, but its lower MIC (minimum inhibitory concentration) for MTX[6] made it a possibly better degrader chassis. We used ΔacrA mutant (Keio JW0452) as an indicative strain since it is clearly MTX-sensitive.

We analysed MXT biotransformation by WT and ΔtolC strains harbouring pSB1C3-cpg2 (BBa_K2688003) or pSB1C3-folC-cpg2 (BBa_K2688009). Strains containing pSB1C3-tet (BBa_R0040) or pSB1C3-folC (BBa_K2688008) were used as controls.

MTX-biotransformation assays

The MTX degradation experiments were performed in LB medium containing 0.5 mM IPTG and 0.2% (m/v) arabinose supplemented with: i) 2% v/v DMSO (which is the solvent we used to prepare MTX stock solution) for the control condition, or ii) with512 µM MTX (kindly provided by Raphaël Labruère).

The degradation experiments were conducted in 15 mL Falcon tubes, with 1 mL of either control or MTX medium, inoculated with the strains at an initial OD of 0.2. The tubes were incubated for 5 to 20 h at 37°C in ambient air. Afterward, the cultures were centrifuged 15 min at 5400 g. The supernatants were filtered (0.22 µM) to eliminate cells and obtain an evaluable supernatant filtrate.

To assess the quantity of MTX remaining, two assays were conducted: HPLC and MTX bioassay.

MTX-detection bioassays

The MTX bioassay was a viability experiment using the ΔacrA indicative strain cultured in 96-wells plate with 50 µL of filtrate and 50 µL ΔacrA (OD 0.04 obtained from an overnight culture in LB).

This viability experiment was conducted in a 96-wells plate over 700 min in a Clariostar® plate reader (BMG LABtech GmbH), with orbital shaking, temperature control at 37°C and ambient air atmosphere, measuring OD600 every 20 min. The transparent plates were sealed with an adhesive transparent top. Duplicate or triplicate were performed for each condition.

HPLC analysis

The HPLC analysis was performed using a reverse phase C18 column. Detection was assessed by a UV spectrophotometer at 303 nm (peak absorption 302-303 nm[7]) with peak-triggered UV-vis spectrum (diode array). The eluting solvent was a gradient of acetonitrile and water (with 0.1% v/v HCOOH). This mode of operation is very similar to the assay and impurities protocols of reference pharmacopeia for MTX. [8][9]




  1. ↑ Rowsell, S., Pauptit, R. A., Tucker, A. D., Melton, R. G., Blow, D. M., & Brick, P. (1997). Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy. Structure (London, England : 1993), 5(3), 337–347. https://doi.org/10.1016/S0969-2126(97)00191-3
  2. ↑ Widemann, B. C., Sung, E., Anderson, L., Salzer, W. L., Balis, F. M., Monitjo, K. S., … Adamson, P. C. (2000). Pharmacokinetics and metabolism of the methotrexate metabolite 2, 4-diamino-N(10)-methylpteroic acid. The Journal of Pharmacology and Experimental Therapeutics, 294(3), 894–901.
  3. ↑ Widemann, B. C., Balis, F. M., Kim, A. R., Boron, M., Jayaprakash, N., Shalabi, A., … Adamson, P. C. (2010). Glucarpidase, leucovorin, and thymidine for high-dose methotrexate-induced renal dysfunction: Clinical and pharmacologic factors affecting outcome. Journal of Clinical Oncology, 28(25), 3979–3986. https://doi.org/10.1200/JCO.2009.25.4540
  4. ↑ Masterson, L. A., Spanswick, V. J., Hartley, J. A., Begent, R. H., Howard, P. W., & Thurston, D. E. (2006). Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy. Bioorganic & Medicinal Chemistry Letters, 16(2), 252–256. https://doi.org/10.1016/J.BMCL.2005.10.017
  5. ↑ Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., … Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Molecular Systems Biology, 2, 2006.0008. https://doi.org/10.1038/msb4100050
  6. ↑ Kopytek, S. J., Dyer, J. C. D., Knapp, G. S., & Hu, J. C. (2000). Resistance to methotrexate due to AcrAB-dependent export from Escherichia coli. Antimicrobial Agents and Chemotherapy, 44(11), 3210–3212. https://doi.org/10.1128/AAC.44.11.3210-3212.2000
  7. ↑ Merck. (2007). The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 14th. Journal of the American Chemical Society, 129(7), 2197–2197. https://doi.org/10.1021/ja069838y
  8. ↑ Cite error: Invalid <ref> tag; no text was provided for refs named PhEur
  9. ↑ Cite error: Invalid <ref> tag; no text was provided for refs named USP-NF

[1]

[2]
  1. ↑ Council of Europe. European Pharmacopoeia. 6th ed. Strasbourg: Council of Europe; 2014. Methotrexate; p. 2735.
  2. ↑ United States Pharmacopeia and National Formulary (USP 30-NF 25). Vol 2. Rockville, MD: United States Pharmacopeial Convention; 2007:1553-1554.