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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
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Glykys, Doris J., Szilvay, Géza R., Tortosa, Pablo, Suárez Diez, María, Jaramillo, Alfonso and Banta, Scott (2011) Pushing the limits of automatic computational protein design : design, expression, and characterization of a large synthetic protein based on a fungal laccase scaffold. Systems and synthetic biology, 5 (1-2). pp. 45-58. doi:10.1007/s11693-011-9080-9 ISSN 1872-5325.
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Official URL: http://dx.doi.org/10.1007/s11693-011-9080-9
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.
Item Type: | Journal Article | ||||||
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Subjects: | Q Science > QD Chemistry Q Science > QK Botany Q Science > QP Physiology T Technology > TP Chemical technology |
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | ||||||
Library of Congress Subject Headings (LCSH): | Laccase, Protein engineering , Protein folding , Globular proteins | ||||||
Journal or Publication Title: | Systems and synthetic biology | ||||||
Publisher: | Springer | ||||||
ISSN: | 1872-5325 | ||||||
Official Date: | June 2011 | ||||||
Dates: |
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Volume: | 5 | ||||||
Number: | 1-2 | ||||||
Number of Pages: | 14 | ||||||
Page Range: | pp. 45-58 | ||||||
DOI: | 10.1007/s11693-011-9080-9 | ||||||
Status: | Peer Reviewed | ||||||
Publication Status: | Published | ||||||
Access rights to Published version: | Restricted or Subscription Access | ||||||
Funder: | Columbia University, Ecole Polytechnique , United States. Air Force. Office of Scientific Research (AFOSR), Merck Research Laboratories, Suomen Akatemia [Academy of Finland], Alfred Kordelinin säätiö [Alfred Kordelin Foundation], Sixth Framework Programme (European Commission) (FP6), Seventh Framework Programme (European Commission) (FP7), Genopole (Firm), Fondation pour la recherche médicale, HPC-Europa programme | ||||||
Grant number: | FA9550-06-1-0264 (AFOSR), NEST-043340 (FP6), ICT-043338 (FP7), KBBE-212894 (FP7), RII3-CT-2003-506079 (HPC) |
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