Team:Freiburg/Methods/Surchem

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Surchem methods

PDITC surface

To enable the detection of an antibody-antigen-interaction in iRIf, the anntigens have to be immobilized on the surface of the iRIf slide. PDITC (p-phenyldiisothiocyanate) can link amino groups through its two isothiocyanate groups and therefore is often used to produce a protein binding surface. To get the PDITC on the surface of the iRIf slides we first activated them with oxygen plasma. This creates very reactive hydroxyl groups on the glass. If a silane is added to the activated glass slide the hydroxyl groups bind to the silicium atoms. By using the silane APTES (3-aminopropyltriethoxysilane) a amino group that can bind the PDITC to the surface is created. With this surface we weren’t only able to immobilize our purified antigens but also to establish a specific Ni-NTA surface on its basis.
Additionally the PDITC chemistry can also be used for the immobilization of DNA. We used this to immobilize the DNA on PDMS (polydimethylsiloxane) slides, which builds the upper part of our two-slides-system.1)

PDITC surface

GOPTS surface

The GOPTS (3-glycidoxypropyltrimethoxysilane) surface is produced in a very similar fashion as the PDITC surface. Through plasma activation reactive hydroxyl groups which bind to later added silanes are created on the glass surface. In contrast to the PDITC surface however the added silane GOPTS, carries an epoxy- instead of an amino group. The epoxy group is a highly strained three atom triangle and therefore very reactive. This allows for the coupling of a variety of different ligands with different functional groups.4) For our application we need to immobilize proteins on the surface of the iRIf slide. This is achieved by the covalent binding of amines, thioles or hydroxy groups of a protein to the epoxy groups on the glass surface. Because there is no need for an additional linker between silane and protein like PDITC the GOPTS surface can be produced much faster. Furthermore GOPTS surfaces normally exhibit less unspecific binding than other silane surfaces due to the formation of a very dense layer.5)
In our experiments the GOPTS surface was able to immobilize proteins, however the PDITC surface showed better binding capacities and worked better as basis for the Ni-NTA/His-tag system, so that we focused on this surface type.

Ni-NTA - His Tag system

The Nickel-NTA (Nitrolotriacetic acid) system is very commonly used for protein purification. Here the coordination of the Imidazole-residue of Histidine to Nickel ions is utilized to bind proteins with a His-tag reversible to Ni-NTA covered columns. The proteins can be eluted with Imidazole which conquers with the Histidine-tag.2) The same coordination can be used to fuse proteins to a surface. We used this to produce specific surfaces on the glass slide of our DiaChip. In our approach we are producing the antigens against which we want to test on demand by cell free expression. After the expression we want only our antigens bound to the surface and not all the other proteins present in the cell free mix. This is why we need a specific surface. All our antigens are cloned with a 10xHis-tag, so they can bind to a Ni-NTA surface.
We managed to establish our own protocol for the preparation of this Ni-NTA surface based on a PDITC surface. On the unspecific PDITC surface NTA with a Lysine-residue (AB-NTA) was immobilized and loaded with Ni-ions, the general setup of the surface is shown in the graphic.3)

Ni-NTA surface

Halo surface

The Halo surface is based on the Halo Tag system developed by Promega. The system consists of a 34 kDa enzyme called the HaloTag that binds covalently to a chloralkane ligand. The binding between this two parts is fast, highly specific and irreversible due to slight modifications of the reactive center of the HaloTag enzyme that prevent dissociation. The drawback of this system is the relatively huge tag, which has to be fused to the protein you want to study. It can interfere with folding, solubility or functionality of the targeted protein.6) 7)
To immobilize proteins specifically on a glass surface we coated the surface with the HaloTag ligand (see picture of halosurface). The ligand is bound to the surface after oxygen plasma activation, as described for PDITC and GOPTS. The HaloTag, which is fused to the protein we want to immobilize, binds to its ligand and therefore preserves the protein of interest on the surface.
We experimented with a variety of different HaloTag ligands, which differed in length of the alkane chain and surface attachment method. The surface consisting of (3-Chloropropyl)triethoxysilane shown in picture x (picture of halosurface from before) showed the most promising results. We were able to immobilize Halo tagged proteins on the surface, but the main challenge to overcome for this surface was minimizing unspecific protein binding. The necessary optimizations, for this surface to be specific enough to be used in our project, could not be performed due to time limitations

Spy surface

The Spy surface is based on the SpyTag/Catcher system. It was created by splitting the CnaB2 domain of the FbaB-protein from Streptococcus pyogenes in two, followed by rational modifications of the fragments. The SpyTag binds to the SpyCatcher through the spontaneous formation of an isopeptide bond within minutes. Due to the covalent nature of this hybridization the binding is irreversible. 8) To immobilize proteins on a surface using the Spy system we tagged the desired protein with the SpyTag. Then the purified SpyCatcher is bound to a protein binding surface like GOPTS or PDITC. Through the binding of SpyTag and SpyCatcher the desired protein is kept at the surface and stably immobilized.
Due to troubles during the cloning and purification of the SpyCatcher we were not able to test the Spy surface.

1) J. Hoffmann et al., 2012. Universal protocol for grafting PCR primers onto various lab-on-a-chip substrates for solid-phase PCR. RCS Adv.
2) J. A. Bornhorst et al., 2000. ] Purification of Proteins Using Polyhistidine Affinity Tags. Methods Enzymol.
3) Y. Asano et al., 2006. Application of an enzyme chip to the microquantiWcation of L-phenylalanine. Anal. Biochem.
4) M. Bañuls et al., 2013. Chemical surface modifications for the development of silicon-based label-free integrated optical (IO) biosensors: A review. Analytica Chimica Acta.
5) J.Phieler et al., 2000. A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces. Biosensors & Bioelectronics.
6) LP Encell et al., 2012. Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Curr Chem Genomics.
7) C. England et al., 2015. HaloTag Technology: A Versatile Platform for Biomedical Applications. Bioconjugate Chem.
8) B. Zakeri et al., 2012. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. Proc Natl Acad Sci U S A.