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Continuous directed evolution of proteins with improved soluble expression.

ABSTRACT: We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield while retaining binding activity. We also developed an activity-independent form of SE-PACE to correct folding-defective variants of maltose-binding protein (MBP) and to evolve variants of the eukaryotic cytidine deaminase APOBEC1 with improved expression properties. These evolved APOBEC1 variants were found to improve the expression and apparent activity of Cas9-derived base editors when used in place of the wild-type cytidine deaminase. Together, these results suggest that SE-PACE can be applied to a wide variety of proteins to rapidly improve their soluble expression.

SUBMITTER: Wang T

PROVIDER: S-EPMC6143403 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Continuous directed evolution of proteins with improved soluble expression.

Wang Tina T Badran Ahmed H AH Huang Tony P TP Liu David R DR

Nature chemical biology 20180820 10

We report the development of soluble expression phage-assisted continuous evolution (SE-PACE), a system for rapidly evolving proteins with increased soluble expression. Through use of a PACE-compatible AND gate that uses a split-intein pIII, SE-PACE enables two simultaneous positive selections to evolve proteins with improved expression while maintaining their desired activities. In as little as three days, SE-PACE evolved several antibody fragments with >5-fold improvement in expression yield w ...[more]

PMID: 30127387

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Project description:The inducible Pm promoter together with its cognate positive transcription regulator XylS has been shown to be useful for recombinant protein production under high cell density conditions. Here we report directed evolution of XylS resulting in mutant proteins with increased ability to stimulate transcription in Escherichia coli from Pm. A first round of mutagenesis using error-prone PCR on xylS was used to construct a library consisting of about 430,000 clones, and this library could be efficiently screened with respect to stimulation of expression from Pm due to a positive correlation between the level of expression of the reporter gene, bla (encoding β-lactamase), and the ampicillin tolerance of the corresponding host cells. Fourteen different amino acid substitutions in XylS were found to separately lead to up to nearly a threefold stimulation of expression under induced conditions, relative to wild type. These mutations were all located in the part corresponding to the N-terminal half of the protein. Varying combinations of the mutations resulted in further stimulation, and the best results (about 10-fold stimulation under induced conditions) were obtained by using a random shuffling procedure followed by a new round of screening. The uninduced levels of expression for the same mutants also increased, but only about four times. Through in silico 3D modelling of the N-terminal domain of XylS, it was observed that the evolved mutant proteins contained substitutions that were positioned in different parts of the predicted structure, including a β-barrel putatively responsible for effector binding and a coiled coil probably important for dimerization. The total production of the host-toxic antibody fragment scFv-phOx expressed from Pm with the evolved XylS mutant protein StEP-13 was about ninefold higher than with wild-type XylS, demonstrating that directed evolution of transcription factors can be an important new tool to achieve high-level recombinant protein production.

Project description:Laboratory evolution has generated many biomolecules with desired properties, but a single round of mutation, gene expression, screening or selection, and replication typically requires days or longer with frequent human intervention. Because evolutionary success is dependent on the total number of rounds performed, a means of performing laboratory evolution continuously and rapidly could dramatically enhance its effectiveness. Although researchers have accelerated individual steps in the evolutionary cycle, the only previous example of continuous directed evolution was the landmark study of Wright and Joyce, who continuously evolved RNA ligase ribozymes with an in vitro replication cycle that unfortunately cannot be easily adapted to other biomolecules. Here we describe a system that enables the continuous directed evolution of gene-encoded molecules that can be linked to protein production in Escherichia coli. During phage-assisted continuous evolution (PACE), evolving genes are transferred from host cell to host cell through a modified bacteriophage life cycle in a manner that is dependent on the activity of interest. Dozens of rounds of evolution can occur in a single day of PACE without human intervention. Using PACE, we evolved T7 RNA polymerase (RNAP) variants that recognize a distinct promoter, initiate transcripts with ATP instead of GTP, and initiate transcripts with CTP. In one example, PACE executed 200 rounds of protein evolution over the course of 8 days. Starting from undetectable activity levels in two of these cases, enzymes with each of the three target activities emerged in less than 1 week of PACE. In all three cases, PACE-evolved polymerase activities exceeded or were comparable to that of the wild-type T7 RNAP on its wild-type promoter, representing improvements of up to several hundred-fold. By greatly accelerating laboratory evolution, PACE may provide solutions to otherwise intractable directed evolution problems and address novel questions about molecular evolution.

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Continuous directed evolution of proteins with improved soluble expression.

Publications

Continuous directed evolution of proteins with improved soluble expression.

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