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Team:Freiburg/Description
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Precipitator
The Concept
We created a cellular, self-replicating purification device for His-tagged proteins. It is a completely artificial fusion protein, which consists of a repeating LRRNT motif domain, coordinating Ni2+ Ions on its surface capped on N and C terminal ends by a hagfish sequence of a similar LRRNT motif. We named this construct “THE PRECIPITATOR”. A second domain, a short hydrophobic peptide stretch, binds a polystyrene surface, called the plastic binding domain. After expression of the Precipitator in a light inducible E. coli strain, the cells are lysed and the lysate is taken up with a serological pipette, in preparation of the actual protein purification steps. The underlying mechanism is comparable to Ni-NTA columns. Our Precipitator protein binds on the surface of the pipette, presenting the chelated Nickel ions. Free coordination sites of the Nickel ions are exposed, so that a His-tagged protein can attach to them. Cells expressing a His-tagged protein can be dissolved by the heat inducible lysis-device. Subsequently, when the lysate is taken up with a serological pipette coated with the Precipitator protein, the His-tagged proteins bind to it. Cell debris is then washed off, while the His-tagged protein stays and is eluted afterwards, in the same fashion as done in Ni-NTA columns with imidazole solutions, increasing in concentration. The His-tagged protein is finally captured in a distinct fraction.
Part design
The Precipitator BBa_K608406 protein is made of an artificial Leucine Rich Repeat (LRR) as the middle part of our own design, capped by C and N-terminal hagfish domain fragments.. This part is one version of three different designed to bind nickel by histidines, grouped together pointing away from the horseshoe shaped protein. Please see modeling for more details
Bacterial LRR Consensus of the central LRR fragment:
LxxLxLxxNxLxxLPxxLPxx
Protein sequence:
CPSRCSCSGTEIRCNSKGLTSVPTGIPSS
ATRLELESNKLQSLPHGVFDK
LTQLTKSNNHLHSLPDNLPAS
LEVLDVSNNHLHSLPDNLPAS
LEVLDVSNNHLHSLPDNLPAS
LEVLDVSNNHLHSLPDNLPAS
LEVLDVSNNHLHSLPDNLPAS
LEVLDVSNNHLHSLPDNLPAS
LKELALDTNQLKSVPDGIFDR
LTSLQKIWLHTNPWDCSCPRIDY
LSRWLNKNSQKEQGSAKCSGSGKPVRSIICP
This protein can be used to complex Nickel or Cobalt. Histidines are positioned in such a way, that they can coordinate the ions from two to four orthogonal oriented directions. Free binding sites of the ions are then exposed, so that a His-tagged protein can attach to them. This protein can be used to complex up to 4 Nickel or Cobalt. The underying design of the protein is of a particular interest, too. LRR are highly conserved motifs throughout evolution. They appear in all kingdoms of life in almost every thinkable role (Ligases, Receptors, Toxins etc.). Their core is highly conserved and provides a very stable backbone, while the non-conserved aminoacids are almost freely interchangeable. Here we investigated an optimal set of non-conserved aminoacids by analysing large sets of similar proteins and databases. You can use this piece of work as a template to design your own protein and give it any function you like, by simply interchanging aminoacids and fusing other domains on the N or C termini. To guarantee proper folding and to shield off the hydrophobic core, a well studied fragment of an LRR protein coming from hagfish was used. This efficiency of this technique was proven before.(REFERENZ). To find out the most likely folding, we designed many different protein sequences, trying out a variety of sets of non coding aminoacids for the LRR and submitted these to the I-TASSER structure prediction
We only submitted one of the three versions, to reduce redundancy in the registry. Please contact us for any questions.
Plastic binding domain
One oft the issues of our project „lab in a cell“ was to use endogenous proteins produced by the cell itself for specific purification and hereby to replace expensive columns. The „precipitator“ designed by our team contains a protein binding domain which complexes nickel and thus enables the binding of His-tagged proteins. After cell lysis the “precipitator” is freely dissolved in the cell lysate. To be able to isolate the “precipitator”-His-tagged-protein-complex from the other cell components it has to be immobilized by another protein domain. The part we designed for this function is the so-called plastic binding domain.
During routine phage display of random peptide libraries, phages were found that bound directly to the plastic surface of the used plastic micro titer plates. The number of plastic binding phages obtained during the phage display experiments depended on the saturation of the plastic micro titer plates with target protein for the antibody-binding phages and could be reduced by the use of blocking proteins as BSA or non-fat milk. Plastic binding phages were resistant to washing steps with PBS alone as well as to PBS in combination with BSA or non-fat dry milk. It was shown that plastic binding phages were even more difficult to recover by acid elution than the “normal” antibody binding phages (Adey et al., 1995).
The binding strength of the plastic binding protein was best on polystyrene plastic surfaces and also observed on PVC-plates (Adey et al., 1995).
The mechanisms by which the phage surface proteins bind to plastic are not well understood. The plastic binding amino acid sequences showed no obvious sequence similarity but where generally enriched by Tyr and Trp residues and are completely devoid of Cys residues. It is possible that the binding comes off non-specific hydrophobic interactions due to partial denaturation of the protein (Cantanero et al., 1980) and potentially due to interactions of the Tyr and Trp residues with the aromatic moieties of the polystyrene plastic surface of micro titer plates.
As well as high hydrophobicity does not necessarily imply plastic binding quality (Menendez et al., 2005) the observed plastic binding phages showed hydrophobic peptide sequences on their surfaces. Expressing such hydrophobic proteins in our host organism E.coli can lead to problems with inclusion bodies or decreased vitality up to cell death.
During our experiments we couldn’t obtain any clones containing both the gene for the plastic binding protein and a constitutive promoter-RBS-construct. We finally succeeded in expressing the plastic binding domain using an inducible promoter. To test the plastic binding domain and get data for our modeling we hereby used an IPTG-inducible promoter. In our completed “Lab in a cell” model the plastic binding domain, as a part of the “precipitator” would be expressed by one of our light inducible promoters.
Green light receptor
Blue light receptor
Red light receptor
Lysis cassette
References
Nils B. Adey et al. 1995 “Characterization of phage that bind plastic from phage-displayed random peptide libraries” Gene 156 (1995) 27-31
Alfredo Menendez & Jamie K. Scott 2005 “The nature of target-unrelated peptides recovered in the screening of phage-displayed random peptide libraries with antibodies” Anal. Biochem. 336 (2005) 145-157
L. A. Cantarero et al. 1980 “The absorptive characteristics of proteins for polystyrene and their significance in solid phase immunoassays” Anal. Biochem. 105 (1980) 375-382
