Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology

Abstract

Artificial metalloenzymes (ArMs) based on the incorporation of a biotinylated metal cofactor within streptavidin (Sav) combine attractive features of both homogeneous and enzymatic catalysts. To speed up their optimization, we present a streamlined protocol for the design, expression, partial purification and screening of Sav libraries. Twenty-eight positions have been subjected to mutagenesis to yield 335 Sav isoforms, which can be expressed in 24-deep-well plates using autoinduction medium. The resulting cell-free extracts (CFEs) typically contain >1 mg of soluble Sav. Two straightforward alternatives are presented, which allow the screening of ArMs using CFEs containing Sav. To produce an artificial transfer hydrogenase, Sav is coupled to a biotinylated three-legged iridium pianostool complex Cp*Ir(Biot-p-L)Cl (the cofactor). To screen Sav variants for this application, you would determine the number of free binding sites, treat them with diamide, incubate them with the cofactor and then perform the reaction with your test compound (the example used in this protocol is 1-phenyl-3,4-dihydroisoquinoline). This process takes 20 d. If you want to perform metathesis reactions, Sav is coupled to a biotinylated second-generation Grubbs-Hoveyda catalyst. In this application, it is best to first immobilize Sav on Sepharose-iminobiotin beads and then perform washing steps. Elution from the beads is achieved in an acidic reaction buffer before incubation with the cofactor. Catalysis using your test compound (in this protocol, 2-(4-(N,N-diallylsulfamoyl)phenyl)-N,N,N-trimethylethan-1-aminium iodide) is performed using the formed metalloenzyme. Screening using this approach takes 19 d.

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Figure 1: Model reactions and structures of the ATHase cofactor [Cp*Ir(biot-p-L)Cl] 3 and the metathesase cofactor 6 (for a synthesis overview see Supplementary Figs. 1 and 2).
Figure 2: Workflow used in this protocol.
Figure 3: Close-up view of the X-ray structure of an artificial transfer hydrogenase based on the biotin-Sav technology.
Figure 4: Summary of designed mutants.
Figure 5
Figure 6: DNA analysis of the plasmid amplification by agarose gel electrophoresis.
Figure 7: Typical calibration curve for Sav K121A using the B4F-assay.

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Acknowledgements

T.R.W. thanks the Swiss National Science Foundation (grants 200020_162348 and the NCCR (National Centres of Competence in Research) Molecular Systems Engineering) and the Seventh Framework Programme Project METACODE (KBBE (Knowledge-Based BioEconomy), 'Code-engineered new-to nature microbial cell factories for novel and safety enhanced bioproduction') and the US National Institutes of Health (NIH; grant GM050781) for generous support. M.H. thanks the SNI (Swiss Nanoscience Institute) for a Ph.D. scholarship. The authors are happy to provide the library free of charge upon request to academic institutions.

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T.R.W. conceived and established the concept of ArMs using the Sav-biotin technology and planned the experiments. H.M. planned experiments, designed the mutant library and established the expression protocol. M.H. and R.R. designed and performed the screening catalysis experiments. T.R.W., H.M., M.H. and R.R. wrote the manuscript.

Corresponding authors

Correspondence to Hendrik Mallin or Thomas R Ward.

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The authors declare no competing financial interests.

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Supplementary Figures 1–7, Supplementary Methods, Supplementary Table 1 and Supplementary Data (PDF 1571 kb)

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Mallin, H., Hestericová, M., Reuter, R. et al. Library design and screening protocol for artificial metalloenzymes based on the biotin-streptavidin technology. Nat Protoc 11, 835–852 (2016). https://doi.org/10.1038/nprot.2016.019

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