Team:Heidelberg/Toolbox Guide/Fusion

From 2014.igem.org

use the intein
TOOLBOX to
ATTACH

First of all, check the DNA sequence of your proteins for EcoRI, XbaI, SpeI, PstI and BsaI sites. If there are E/X/S/P sites, you might have problems to change your backbone or add a promotor. If there is a BsaI site, the cloning will be more difficult.

At which position do you want to add your posttranslational modification?

Does your protein of interest have methionin as a first amino acid?

Protocol

  • Get BBa_K1362141 (RBS + SspDnaB C-intein RFC [] assembly construct) from the registry of standard biological parts. Get BBa_K1362110 (RBS + SspDnaX-S11 N-intein RFC [] assembly construct) from the registry of standard biological parts.

  • Divide your protein of interest into a short N-terminal part including the synthetic attachment and a long C-terminal one. a long N-terminal part including the synthetic attachment and a short C-terminal one.

  • Get the DNA of the long part of your protein of interest.

  • The long part of your protein of interest will be expressed recombinantly. Therefore design and order primers for this part:

    • In 5'-3' direction, the forward primer should have the following sequence:

      NNNNNNGGTCTCCCAACAGCATTCGCAGC+ binding part NNNNNNGGTCTCCGATG + binding part NNNNNNGGTCTCCG + binding part

    • In 5'-3' direction, the reverse primer should have the following sequence:

      NNNNNNGGTCTCTATTA + binding part NNNNNNGGTCTCGAGCAACCAGATTCACGCAG + binding part

  • Use PCR to create your insert and purify it.

  • Use Golden Gate assembly to insert the recombinant part of your protein of interest into BBa_K1362141 (SspDnaB C-intein RFC [???] assembly construct) BBa_K1362110 (SspDnaX N-intein RFC [???] assembly construct)

    Since there is a BsaI site in your protein, you have to religate after the Golden Gate reaction.

  • Induce expression.

  • Purify the protein.

  • Synthesize the short part of your protein including the posttranslational modification together with the split intein sequence.

    The sequence should look like this:

    N-terminal protein part with modification - SEFSGCISGDSLISLA GLLVHNCHT - C-terminal protein part with modification

  • Mix both parts in vitro to induce protein trans-splicing.

    Further information about this step can be found here:

    Mootz, Henning: Split Inteins as Versatile Tools for Protein Semisynthesis. ChemBioChem 2009, 10, 2579 - 2589 . DOI: 10.1002/cbic.200900370 .

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