Difference between revisions of "Part:BBa K4275010"
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[[File:GreatBay SCIE--3D MHETase-t.png|950px]]
 
[[File:GreatBay SCIE--3D MHETase-t.png|950px]]
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<p align="center"><b>Figure 1</b> The 3D structure of the protein predicted by Alphafold2. </p>
  
 
===Usage and Biology===
 
===Usage and Biology===
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The artificially-designed MHETase - Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K.marxianus via ScGPI, or binds to E. coli's Cell-surface Nanobody3(Nb3). It is believed that the immobilization of the two enzymes (FAST-PETase-t and MHETase-t) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.
 
  
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The artificially-designed MHETase - Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of <i>K. marxianus</i> via ScGPI, or binds to <i>E.coli</i>'s Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (PETase-5-dockerin and MHETase-t) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.
  
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===Design consideration===
 
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1. The sequence of the first 565 amino acids is identical with the free MHETase (BBa_K4275009).
 
  
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2. A 10 aa long GS linker (GGGGS)2 is appended following the Pro565 residue at the C' terminal of the original sequence.
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===Sequence and Features===
  
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3. The 79 amino acid long type-I dockerin domain is fused at the terminal of the GS linker, followed by a TEV site and a 8xhis affinity purification tag (HHHHHHHH).
 
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4. DNA sequence is codon-optimized based on the codon-usage table of E.Coli Strain K12.MG1655.
 
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K4275010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4275010 SequenceAndFeatures</partinfo>
  

Revision as of 23:51, 11 October 2022


MHETase-t

MHETase-t is a dockerin-fused variant of free MHETase (BBa_K4275009). The enzyme shares all the common sequences in the CDS with the former, except the C' terminal fusion of a type-I dockerin domain in this enzyme. The catalytic domain of MHETase-t and the dockerin domain are interspaced with a medium-lengthed flexible GS linker (10 aa long) to avoid steric inhibitions. The type-I dockerin domain forms high-affinity non-covalent interaction with type-I cohesins on the cipA scaffoldin.

GreatBay SCIE--3D MHETase-t.png

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

The artificially-designed MHETase - Dockerin I fusion protein could be tightly-anchored onto the CipA scaffoldin via the high-affinity Doc I: Coh I noncovalent interaction. The CipA primary scaffoldin is then tightly-anchored onto the secondary scaffoldin - OlpB, which is either anchored onto the cell surface of K. marxianus via ScGPI, or binds to E.coli's Cell-surface Nanobody3(Nb3)(BBa_K4275026). It is believed that the immobilization of the two enzymes (PETase-5-dockerin and MHETase-t) could increase their proximity and further enhance their synergy, whilst the affinity of carbohydrate-binding module 3 (CBM3) on the CipA scaffoldin towards PET fiber could further increase the catalytic efficiency of this degradation complex.


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 123
    Illegal NgoMIV site found at 237
    Illegal NgoMIV site found at 693
    Illegal AgeI site found at 190
    Illegal AgeI site found at 307
  • 1000
    COMPATIBLE WITH RFC[1000]