Team:Heidelberg/pages/Toolbox

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<a href="https://2014.igem.org/Team:Heidelberg/Toolbox/Circularization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/5/58/Heidelberg_Toolbox_Circularization.png" class="img-responsive" /></span>CIRCULARIZE</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Oligomerization"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/4/40/Oligomerization.png" class="img-responsive" /></span>OLIGOMERIZE</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Fusion_and_Tagging"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/8/87/Heidelberg_Toolbox_Fusion.png" class="img-responsive" /></span>FUSE & TAG</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#Purification"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/0/04/Heidelberg_Toolbox_Purification.png" class="img-responsive" /></span>PURIFY</a>
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<a href="https://2014.igem.org/Team:Heidelberg/Project/Toolbox#On_Off"><span class="col-lg-offset-3 col-lg-6 col-md-offset-2 col-md-8 col-sm-12 nopadding nofloat"><img src="/wiki/images/c/c2/Heidelberg_Toolbox_On-Off.png" class="img-responsive" /></span>ACTIVATE &<br />DEACTIVATE</a>
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=Introduction=
=Introduction=

Revision as of 22:20, 16 October 2014

CIRCULARIZE
OLIGOMERIZE
FUSE & TAG
PURIFY
ACTIVATE &
DEACTIVATE

Contents

Introduction

Placeholder

Circularization

Millions of years of evolution have allowed proteins to performed extremely specific chemical modifications that are not only essential for living organisms but can also be of great benefit to produce useful molecules for our life, efficiently and at low cost. A major limitation of the use of enzyme for industrial application and in general for usage out of their natural environment is their stability. They can be destroyed by other enzymes and they can unfold and take non-functional conformation when exposed to non-physiological temperature and pH. Such limitations has motivated research in species that can grow at extreme temperatures [6]. Another major area of chemical research if the design of strategies to stabilize enzymes, and more generally proteins and peptides. Protein circularization, meaning ligation of the N- and C-terminal ends of a protein, represents a promising way to achieve this stabilization. While conserving the functionality of their linear counterpart, circular proteins can be superior to linear proteins in terms of thermostability [1][2][3], resistance against chemical denaturation [4] and protection from exopeptidases [2][4]. Moreover, a circular backbone can improve in vivo stability of therapeutical proteins and peptides [5]. These remarkable properties motivated us to develop new tools to circularize any protein of interest.

Our Toolbox Guide provides a step-by-step strategy to clone a circularization linker and express it in e. Coli. Moreover, in case of complex structures where the protein extremities are far from each other, we have developed a software that will design the appropriate rigid linkers.

Oligomerization

Split inteins constitute a useful tool to produce huge polymers in vivo: Hauptmann et al. managed to fabricate synthetic spider silk with microfiber structure. The results using an easy-to-handle split intein system were stunning: The polymers had a molecular weight of 250 kDa and more.[1]

Standardization of a unique tool

The valuable properties of spider silk, for example its exceptional strength and elasticity, result from numerous repeats of certain protein motifs. Convenitonal methods to multimerize these motifs bear a lot of difficulties: Often genetic and mRNA instability constitute a barrier for the production of multimers as fusion proteins.[1] Posttranslational assembly through split inteins is therefore the solution to overcome these problems. The successfull polimerization of spider silk potein motifs demonstrates the potential of split inteins to be a useful tool for the production of new biomaterials by performing oligomerization reactions with split inteins. The iGEM team Heidelberg standardized (lik to toolbox guide) the oligomerization procedure with split inteins to allow easy handling with different proteins.

The mechanism

Oligomerization reactions require the same constructs as the ones used for protein circularization.

§§§image§§§

Circularization is achieved by bringing the N and C terminus of a protein very close together, so both intein parts can asseble, cut out off the protein and thereby circularize it. In contrast, oligomerization occurs when the both termini of a protein cannot reach each other and the intein parts of two neighbouring proteins assemble, at the same assembling the protein parts.

§§§image§§§

References

[1] Hauptmann, V. et al.: Native-sized spider silk proteins synthesized in planta via intein-based multimerization. Transgenic Res (2013) 22:369–377. DOI 10.1007/s11248-012-9655-6.

Fusion and Tagging

Placeholder

Purification

Split-inteins can be used to simplify conventional protein purification in many aspects: a split-intein based system is cheaper, faster and more efficient than other purification procedures. In fact, only a single chromatographic step is necessary for both, binding of the protein fused to an affinity tag to the affinity column and tag removal. Also, there is no need for expensive proteases. We standardized this split-intein purification system, so it is easy applicable to all kinds of proteins.

In previous purification procedures, there a several steps to take to get solely the purified protein: The first step is the binding of the protein to the affinity matrix, the washing and finally release of the protein from the affinity column, the protein still being bound to the affinity tag. The next step is the removal of the affinity tag from the protein by protease cleavage, which causes undesirable secondary cleavage within the protein sequence. In industrial scales proteases are the most expensive component during the purification process. Finally, the split off affinity tag has to be separated from the protein, which takes again a lot of effort.

The split-intein based system is manifold easier: All these steps can be combined into one by using Ssp DnaB mini-intein, an artificial split intein. It performs the trans-splicing reaction only under certain pH and temperature conditions. A mutation of the SspDnaB N-intein at Cys1 to Ala prevents cleavage at the N-terminus, cleavage at the C-terminus still occurs. The N-terminal intein part, attached to a chitin binding domain can be used as an affinity ligand, while the C-terminal part, fused to a protein of interest, can be used as an affinity tag binding highly specific to the ligand part. After loading the protein of interest on a chitin column, the trans-splicing reaction can be induced by adjusting temperature and pH: the both split inteins fuse together, disrupting the fusion to the protein of interest in one single step [1].

References

[1] Lu, Wei et al.: Split intein facilitated tag affinity purification for recombinant proteins with controllable tag removal by inducible auto-cleavage. J. Chromatogr. A 1218 (2011) 2553–2560. DOI: 10.1016/j.chroma.2011.02.053.

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