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Part:BBa_K3204001

Designed by: Nikola Vinko   Group: iGEM19_NAWI_Graz   (2019-09-16)
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C-LytAm7 Affinity Polypeptide Tag

Usage and Biology

The C-LytAm7 is a modified C-LytA polypeptide which is a C-terminal domain of the LytA amidase from Streptococcus pneumoniae that binds to choline residues present in the cell wall of Streptococcus pneumoniae. The recognition of the choline residues and analogs has allowed the development of an efficient system for the overexpression and purification of fusion proteins tagged to C-LytA using commercial resins such as diethylaminoethanol (DEAE) cellulose or even aqueous two-phase systems[1].

The use of relatively sizeable tags such as C-LytA (136 aa) may help the immobilized protein of interest to avoid steric hindrances with the support and to increase its mobility. Additionally, the C-LytAm7 shows higher kinetic stability and higher denaturation temperatures (up to 90°C) than the wild-type C-LytA[2].

C-LytA is also being used in construction of enzymatic electrochemical cells and biosensors (gold or graphite electrodes) that can be easily regenerated and reused after the eventual enzyme inactivation simply by washing the electrode with choline and reloading with a fresh protein. The C-LytA system presents some advantages over other affinity tags, such as a higher buffer compatibility, non-interference with metalloproteins, a complete reversibility of binding the possibility of employing tailor-made supports with a wide range of compounds to choose from.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Characterization

Overview

  • The overexpression and purification system of the modified polypeptide version C-LytAm7 has been tested.

Overexpression and Purification of C-LytAm7 Polypeptide

E. coli cells with a C-LytAm7 containing plasmid were grown in 3x 800 mL LB Broth cultures, 140 rpm, overnight. The cultures were then induced with IPTG in order to induce the protein expression. A 1 mL aliquot was taken before the induction as a control sample. The cells were then harvested, sonicated and applied on a DEAE-Sepharose Fast-flow column (20 mL). The column was washed first with five column volumes of ice-cold 0.05 M Tris-maleate buffer, pH 7.0. Second wash was done with five column volumes of the same wash buffer plus 1.5 M NaCl, and then eluted with the ice-cold 0.05 M Tris-maleate buffer, pH 7.0, 1.5 M NaCl, 2 % choline buffer. Fractions were analyzed for homogeneity by SDS-PAGE[3].


Figure 1: Ion-Exchange Chromatography of E.coli total extract on DEAE-Sepharose column and assessment of fraction homogeneity by SDS-PAGE. Lane 1 shows a PageRuler™ Prestained Protein Ladder, 10 to 180 kDa; lanes FT1 and FT2 show the flowthrough; lanes W1 and W2 show the first and second wash fractions, respectively; Control shows the uninduced E.coli samples; lanes E1-E4 show the elution fractions.

Conclusion

  • The SDS-PAGE shows that the overexpression and purification of the C-LytAm7 (15 kDA) yielded high amount of protein with high protein purity.


References

1. Sánchez-Puelles, JoséM., Sanz, J. M., Garcia, JoséL. & Garcia, E. Cloning and expression of gene fragments encoding the choline-binding domain of pneumococcal murein hydrolases. Gene 89, 69–75 (1990).
2. Hernandez-Rocamora, V. M., Maestro, B., Molla-Morales, A. & Sanz, J. M. Rational stabilization of the C-LytA affinity tag by protein engineering. Protein Engineering Design and Selection 21, 709–720 (2008).
3. Bello-Gil, D. et al. Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes. PLoS One 9, (2014).



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