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Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold

  • METHODOLOGY ARTICLE
  • Published:
Systems and Synthetic Biology

Abstract

The de novo engineering of new proteins will allow the design of complex systems in synthetic biology. But the design of large proteins is very challenging due to the large combinatorial sequence space to be explored and the lack of a suitable selection system to guide the evolution and optimization. One way to approach this challenge is to use computational design methods based on the current crystallographic data and on molecular mechanics. We have used a laccase protein fold as a scaffold to design a new protein sequence that would adopt a 3D conformation in solution similar to a wild-type protein, the Trametes versicolor (TvL) fungal laccase. Laccases are multi-copper oxidases that find utility in a variety of industrial applications. The laccases with highest activity and redox potential are generally secreted fungal glycoproteins. Prokaryotic laccases have been identified with some desirable features, but they often exhibit low redox potentials. The designed sequence (DLac) shares a 50% sequence identity to the original TvL protein. The new DLac gene was overexpressed in E. coli and the majority of the protein was found in inclusion bodies. Both soluble protein and refolded insoluble protein were purified, and their identity was verified by mass spectrometry. Neither protein exhibited the characteristic T1 copper absorbance, neither bound copper by atomic absorption, and neither was active using a variety of laccase substrates over a range of pH values. Circular dichroism spectroscopy studies suggest that the DLac protein adopts a molten globule structure that is similar to the denatured and refolded native fungal TvL protein, which is significantly different from the natively secreted fungal protein. Taken together, these results indicate that the computationally designed DLac expressed in E. coli is unable to utilize the same folding pathway that is used in the expression of the parent TvL protein or the prokaryotic laccases. This sequence can be used going forward to help elucidate the sequence requirements needed for prokaryotic multi-copper oxidase expression.

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Acknowledgments

The authors would like to acknowledge the financial support of a Joint Research Project Award from the Alliance Program involving Columbia University and École Polytechnique awarded to S. B and A. J. Financial support was also provided by an AFOSR MURI award (FA9550-06-1-0264) to S. B. D. J. G. acknowledges support from Merck & Co., Inc. and G. S. Z. from the Academy of Finland and the Alfred Kordelin Foundation. A. J. acknowledges support from FP6-NEST-043340 (BioModularH2), FP7-ICT-043338 (Bactocom), FP7-KBBE-212894 (Tarpol), the ATIGE-Genopole and the Fondation pour la Recherche Medicale. A. J. also acknowledges the HPC-Europa program (RII3-CT-2003-506079) and the BSC for supercomputing time. The authors also thank Dr. Ian Wheeldon for the expression and purification of the SLAC protein.

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Authors and Affiliations

  1. Department of Chemical Engineering, Columbia University in the City of New York, 820 S. W. Mudd, MC 4721, 500 W 120th St, New York, NY, 10027, USA

    Doris J. Glykys, Géza R. Szilvay & Scott Banta

  2. ISSB, Genopole-Université d’Évry Val d’Essonne-CNRS UPS3201, Batiment Geneavenir 6. 5, rue Henri Desbruères., 91030, Evry Cedex, France

    Pablo Tortosa, María Suárez Diez & Alfonso Jaramillo

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  1. Doris J. Glykys
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  2. Géza R. Szilvay
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  3. Pablo Tortosa
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  4. María Suárez Diez
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  5. Alfonso Jaramillo
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  6. Scott Banta
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Correspondence to Scott Banta.

Electronic supplementary material

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11693_2011_9080_MOESM1_ESM.doc

The supplementary material available in the electronic edition contains the DNA sequence of the DLac gene, buffers and substrates used in the kinetics measurements, and a summary of the mass spectrometry results for the DLac proteins (Glykys et al. Supp Mat.doc) A PDB file of the modeled DLac protein is also included in Supplementary Material. (DOC 88 kb)

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Glykys, D.J., Szilvay, G.R., Tortosa, P. et al. Pushing the limits of automatic computational protein design: design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold. Syst Synth Biol 5, 45–58 (2011). https://doi.org/10.1007/s11693-011-9080-9

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  • DOI: https://doi.org/10.1007/s11693-011-9080-9

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