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Expression and In Vivo Loading of De Novo Proteins with Tetrapyrrole Cofactors

Part of the Methods in Molecular Biology book series (MIMB,volume 2397)


Tetrapyrrole cofactors such as heme and chlorophyll imprint their intrinsic reactivity and properties on a multitude of natural proteins and enzymes, and there is much interest in exploiting their functional and catalytic capabilities within minimal, de novo designed protein scaffolds. Here we describe how, using only natural biosynthetic and post-translational modification pathways, de novo designed soluble and hydrophobic proteins can be equipped with tetrapyrrole cofactors within living Escherichia coli cells. We provide strategies to achieve covalent and non-covalent heme incorporation within the de novo proteins and describe how the heme biosynthetic pathway can be co-opted to produce the light sensitive zinc protoporphyrin IX for loading into proteins in vivo. In addition, we describe the imaging of hydrophobic proteins and cofactor-rich protein droplets by electron and fluorescence microscopy, and how cofactors can be stripped from the de novo proteins to aid in vitro identification.

Key words

  • De novo proteins
  • Tetrapyrroles
  • In vivo cofactor loading
  • Fluorescence imaging
  • Electron microscopy
  • Heme
  • Protein expression and purification

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The authors wish to thank Lorna Hodgson and Paul Verkade for their support with the methods for cell imaging. This work was supported at the University of Bristol by the BBSRC (grant no: BBI014063/1, BB/R016445/1 and BB/M025624/1).

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Correspondence to J. L. Ross Anderson .

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Curnow, P., Anderson, J.L.R. (2022). Expression and In Vivo Loading of De Novo Proteins with Tetrapyrrole Cofactors. In: Magnani, F., Marabelli, C., Paradisi, F. (eds) Enzyme Engineering. Methods in Molecular Biology, vol 2397. Humana, New York, NY.

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