Team:TU-Munich/Project/Localisation

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Localization in Physcomitrella patens

Figure 1: Overview of different possible localisations for effector proteins.

In order to use Physcomitrella as a chassis for Phytoremediaton, it is essential to be able to express effector proteins in different compatiments. This includes cytoplasmatic expression of cytosolic effectors which degrade xenobiotics capable of crossing the cell membrane and which might depend on cofactors for degradation or conjugation. Secondly, there is the possibility to secrete effectors outside of the cell for an easy access to their respective target molecules. Other applications benefit of the expression of immobilized effectors on the inner or outer cellular membrane. This allowing the creation of systems that dont release transgenic proteins into the environment and of systems able to internalize substances attached by recombinant binding proteins.

Cytosolic expression of effector proteins is achieved by cloning the respective BioBrick downstream of the Actin_5 promoter in either RFC 10 or RFC 25. The secretion of effectors is achieved through the addition of signal peptides of an antibody [Fussenegger] and also tested the signal peptide from the SERK receptor of Physcomitrella patens which both should be suitable BioBricks to accomplish secretion. For this purpose the signal peptides were created as RFC 25 N-parts and the effectors need to be availible in RFC 25 to create fusion proteins. Finally the inclusion of recombinant effector proteins into a receptor which is functional in Physcomitrella patens was investigated by the construction of a synthetic receptor based on the SERK receptor

Cytoplasmatic Expression

Cytoplasmatic expression of effector proteins is necessary, if the effector either is dependent from cofactors that only exist in the intracellular milieu or its mechanism is connected to at least one intracellular component which does not appear outside of the cell. In our case, it would be:

Catechol-1,2-dioxygenase: needs ferredoxins to regenerate the oxygen-inactivated iron

Glutathion-S-transferase: needs glutathion for coupling reactions

Secretory Expression

If some effectors proteins contain disulfid bonds, that effector has to be secreted as the cytosolic compartment does not allow formation of disulfid bonds due to its reducing milieu. For secretion, that protein has be fused to a signal peptide for translocation into the endoplasmatic reticulum. For this purpose we created two biobricks that codes for a signal sequence for endoplasmatic reticular translocation, namely SERK signal peptide ([http://parts.igem.org/Part:BBa_K1159303 BBa_K1159303]) of the Somatic Embryogenesis Receptor Kinase from Physcomitrella patens and the IgKappa signal peptdie of the Ig Kappa chain from Mus musculus ([http://parts.igem.org/Part:BBa_K1159304 BBa_K1159304]). In our case, following effector should be secreted:

Laccase from Bacillus pumilus: has 1 disulfid bond

Membrane-bound Expression

Figure x:

For substance binding proteins or as a alternative to secretion for degrading enzymes, it would be convenient to anchor the effectors on the surface of the plant, namely the cytoplasmatic membrane.

For this purpose, we have not only to add a signal for secretion at the N-terminus of our effector protein, but also have to add a downstream transmembrane domain for membrane localization. Furthermore, we fuse GFP to the intracellular site of the transmembrane region in order to verify membrane localization by fluorescence microscopy. Between the effector protein and the transmembrane region, we either add a Strep-tag II and a TEV cleavage site; so one can characterize the membrane-bound effectors by only has to incubating the plant with a TEV Protease resulting in a release of the membrane bound effectors and afterwards can purify them through the Strep-tag II.

Advanced expression: Employing SpyTag & SpyCatcher for post-translational fusion

Figure x::

At a certain point it will be necessary to think about transgenic moss which is able to degrade several different xenobiotics using different effector molecules. In order to prepare for these posterior needs we decided to integrate the SpyCatcher and SpyTag system into our project [Ref]. This Spy-system allows for the creation of posttranslational protein fusion based on a covalent bond which is formed between the side chains of residues of a SypCatcher and a SpyTag.
Therefore it is possible to create a protein fusion of the SypTag with a recombinant effector protein which is expressed separately (e.g. with anothe expression strength) and becomes in the secretory pathway fused to a receptor which contains the SypCatcher. By these means it becomes possible to express the SERK-receptor under a strong promoter and to adjust the expression of different effector proteins to their particuluar necessity (Fig. x B).
On the other hand enzymatic effectors might be active as multimeric proteins and thus not every subunit can be fused to a receptor for steric reasons. In this case the application of the SpyTag system also seems advantageous as it allows the multimeric protein to assemble into its functional form before it becomes immobilized to the outer side of the cellular membrane by its SpyTags (Fig. x C).

References:

http://www.plantphysiol.org/content/127/4/1430 Schaefer and Zryd, 2001 Schaefer, D.G. and Zrÿd, J. (2001). The Moss Physcomitrella patens, Now and Then. Plant Physiology, 127(4):1430-1438.

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 == Localization in ''Physcomitrella patens'' ==
-[[File:TUM13_Localization.png|thumb|center|910px| Figure x:]]
+[[File:TUM13_Localization.png|thumb|center|910px| Figure 1: Overview of different possible localisations for effector proteins.]]
 In order to use ''Physcomitrella'' as a chassis for Phytoremediaton, it is essential to be able to express effector proteins in different compatiments. This includes cytoplasmatic expression of cytosolic effectors which degrade xenobiotics capable of crossing the cell membrane and which might depend on cofactors for degradation or conjugation.  Secondly, there is the possibility to secrete effectors outside of the cell for an easy access to their respective target molecules. Other applications benefit of the expression of immobilized effectors on the inner or outer cellular membrane. This allowing the creation of systems that dont release transgenic proteins into the environment and of systems able to internalize substances attached by recombinant binding proteins.<br>