Difference between revisions of "Part:BBa K5121013"

 
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<partinfo>BBa_K5121013 short</partinfo>
 
<partinfo>BBa_K5121013 short</partinfo>
  
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== Biology ==
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===Usage and Biology===
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RebA encodes an 18 kDa protein which forms one of the main structural components of type 51 R bodies. This part is based off of the wildtype rebA part — BBa_K2912000 — uploaded by SZU-China in 2019, however has been modified to be compatible with cysteine maleimide conjugation at the N-terminus. This is to allow for conjugation with entire R bodies — note that R bodies should be assembled and purified before conjugation with the constituent rebA monomers. Our team aimed to use conjugation to attach drugs onto R bodies for use in drug delivery.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K5121013 SequenceAndFeatures</partinfo>
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Cysteine maleimide conjugation is a form of Michael addition, in which the thiol of the cysteine acts as a nucleophile to react with maleimide, forming a thiosuccinimide adduct (Figure 1). Through this reaction, drugs with maleimide groups can hence be reacted onto proteins with readily accessible cysteines.
  
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===Functional Parameters===
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<partinfo>BBa_K5121013 parameters</partinfo>
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    <img src="https://static.igem.wiki/teams/5121/rebcm/screenshot-2024-09-30-at-12-07-08-am.png" width="80%">
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    <figcaption>
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      Figure 1. Cysteine maleimide conjugation reaction mechanism.
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Since wildtype rebA does not have any cysteines (and nor does RebB), maleimide conjugation provides an ideal conjugation method for orthogonal attachment on to R bodies. This part contains an N-terminal cysteine that has been added to facilitate N-terminal conjugation using cysteine maleimide addition. See the design page for more details.
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<span class='h3bb'><u>Sequence and Features</u></span>
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<partinfo>BBa_K5121027 SequenceAndFeatures</partinfo>
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Revision as of 12:18, 2 October 2024


rebA N-terminal CGGGGS

Biology

RebA encodes an 18 kDa protein which forms one of the main structural components of type 51 R bodies. This part is based off of the wildtype rebA part — BBa_K2912000 — uploaded by SZU-China in 2019, however has been modified to be compatible with cysteine maleimide conjugation at the N-terminus. This is to allow for conjugation with entire R bodies — note that R bodies should be assembled and purified before conjugation with the constituent rebA monomers. Our team aimed to use conjugation to attach drugs onto R bodies for use in drug delivery.

Cysteine maleimide conjugation is a form of Michael addition, in which the thiol of the cysteine acts as a nucleophile to react with maleimide, forming a thiosuccinimide adduct (Figure 1). Through this reaction, drugs with maleimide groups can hence be reacted onto proteins with readily accessible cysteines.

Figure 1. Cysteine maleimide conjugation reaction mechanism.

Since wildtype rebA does not have any cysteines (and nor does RebB), maleimide conjugation provides an ideal conjugation method for orthogonal attachment on to R bodies. This part contains an N-terminal cysteine that has been added to facilitate N-terminal conjugation using cysteine maleimide addition. See the design page for more details.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 803
    Illegal NheI site found at 865
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 745
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]