pubmed.ncbi.nlm.nih.gov

Phage-assisted continuous evolution of proteases with altered substrate specificity - PubMed

  • ️Sun Jan 01 2017

Phage-assisted continuous evolution of proteases with altered substrate specificity

Michael S Packer et al. Nat Commun. 2017.

Abstract

Here we perform phage-assisted continuous evolution (PACE) of TEV protease, which canonically cleaves ENLYFQS, to cleave a very different target sequence, HPLVGHM, that is present in human IL-23. A protease emerging from ∼2500 generations of PACE contains 20 non-silent mutations, cleaves human IL-23 at the target peptide bond, and when pre-mixed with IL-23 in primary cultures of murine splenocytes inhibits IL-23-mediated immune signaling. We characterize the substrate specificity of this evolved enzyme, revealing shifted and broadened specificity changes at the six positions in which the target amino acid sequence differed. Mutational dissection and additional protease specificity profiling reveal the molecular basis of some of these changes. This work establishes the capability of changing the substrate specificity of a protease at many positions in a practical time scale and provides a foundation for the development of custom proteases that catalytically alter or destroy target proteins for biotechnological and therapeutic applications.Proteases are promising therapeutics to treat diseases such as hemophilia which are due to endogenous protease deficiency. Here the authors use phage-assisted continuous evolution to evolve a variant TEV protease with altered target peptide sequence specificities.

PubMed Disclaimer

Conflict of interest statement

The authors have filed provisional patent applications on PACE technologies and on the evolved gene products that are disclosed in this manuscript..

Figures

Fig. 1
Fig. 1

Overview of protease PACE. A culture of host E. coli continuously dilutes a fixed-volume vessel containing an evolving population of selection phage (SP) in which essential phage gIII has been replaced by a protease gene. These host cells contain an arabinose-inducible mutagenesis plasmid (MP) and an accessory plasmid (AP) that supplies gIII. The expression of gIII is made protease-dependent through the use of a protease-activated RNA polymerase (PA-RNAP) consisting of T7 RNA polymerase fused through a cleavable substrate linker to T7 lysozyme, a natural inhibitor of T7 RNAP transcription. If an SP encodes a protease capable of cleaving the substrate linker, then the resulting liberation of T7 RNAP leads to the production of pIII and infectious progeny phage encoding active proteases. Conversely, SP encoding proteases that cannot cleave the PA-RNAP yield non-infectious progeny phage

Fig. 2
Fig. 2

PACE evolutionary trajectories. Across the eight stages of PACE along three diverging trajectories (shown in purple, blue, and orange), each arrow represents a PACE experiment with the corresponding substrate peptide and selection stringency parameters listed beneath the arrow. Increased selection stringency annotations are: Q649S (a T7 RNAP mutant with decreased transcriptional activity), proA (lower expression of substrate PA-RNAP), and IL-23 (38–66) (native IL-23 sequence in place of GGS linker). Numbers above the arrows denote TEV protease residues that were targeted in site-saturation mutagenesis libraries used to initiate that PACE experiment. In the first PACE experiment, wild-type TEV protease was mutagenized at the positions shown. All other libraries were generated using the protease genes emerging from the previous PACE stage as the PCR template. For PACE stages with no targeted mutagenesis, lagoons were inoculated with an aliquot of the phage population from the preceding experiment

Fig. 3
Fig. 3

Protease specificity profiling. a Overview of phage substrate display. M13 bacteriophage libraries encode pIII fused to a FLAG-tag through a randomized protease substrate linker. These substrate phage are bound to anti-FLAG magnetic beads and treated with a protease to release phage that encode substrates that can be cleaved by the protease. The remaining intact substrate phage are eluted with excess FLAG peptide. The abundance of all substrate sequences within the cleaved and eluted samples is measured by high-throughput sequencing. b–e For all assayed proteases, phage substrate display was separately performed on seven libraries, each with a different single randomized position within the ENLYFQS motif. The results are displayed as sequence logos, with letter height proportional to enrichment in the cleaved versus eluted sample. Letters placed above the x-axis indicate protease acceptance and letters beneath the axis indicate rejection. b Wild-type TEV protease exhibits strong enrichment for the consensus motif EXLYFQS. c Evolved TEV L2F has broadened specificity at P6 and shifted specificity at P3, P1, and P1ʹ in accordance with the HPLVGHM target substrate. d Mutations I138T, N171D, and N176T are sufficient to broaden P6 specificity. e Mutations T146S, D148P, S153N, S170A, and N177M shift specificity at both P1 and P3

Fig. 4
Fig. 4

Protease-mediated attenuation of IL-17 secretion in mouse splenocytes. The activity of IL-23 in vivo is mediated by stabilization of a T-helper cell lineage (Th17) that secretes IL-17, leading to downstream pro-inflammatory signals. This pathway can be assayed within a culture of mouse mononuclear splenocytes, by measuring the amount of IL-17 secretion into the cell culture media using an ELISA. As a positive control, anti-IL-23 antibodies in a super-stoichiometric ratio prevent IL-17 secretion. Preincubation of IL-23 and evolved TEV L2F attenuates IL-17 secretion, demonstrating that cleavage of the HPLVGHM target site inactivates immune signaling capabilities of IL-23. Values represent the mean and error bars represent the standard deviation of three technical replicates

Similar articles

Cited by

References

    1. Rawlings ND, Barrett AJ, Finn R. Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors. Nucleic Acids Res. 2016;44:D343–D350. doi: 10.1093/nar/gkv1118. - DOI - PMC - PubMed
    1. Craik CS, Page MJ, Madison EL. Proteases as therapeutics. Biochem. J. 2011;435:1–16. doi: 10.1042/BJ20100965. - DOI - PMC - PubMed
    1. Zhao HM, Arnold FH. Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng. 1999;12:47–53. doi: 10.1093/protein/12.1.47. - DOI - PubMed
    1. You L, Arnold FH. Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide. Protein Eng. 1996;9:77–83. doi: 10.1093/protein/9.1.77. - DOI - PubMed
    1. Persson E, Kjalke M, Olsen OH. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proc. Natl Acad. Sci. USA. 2001;98:13583–13588. doi: 10.1073/pnas.241339498. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances