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Development of directed-evolution methods utilizing combinatorial protein libraries in Escherichia coli

Time: Tue 2024-06-04 13.00

Location: F3 (Flodis), Lindstedtsvägen 26 & 28, Stockholm

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Language: English

Subject area: Biotechnology

Doctoral student: Luke Parks , Proteinvetenskap

Opponent: Professor Ola Söderberg, Department of Pharmaceutical Biosciences, Uppsala University, Sweden

Supervisor: Professor John Löfblom, Proteinvetenskap; Professor Stefan Ståhl, Proteinvetenskap

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QC 2024-05-06


Directed evolution using combinatorial protein libraries is a tremendously powerful technique for the generation of proteins with new or improved properties. A key aspect in such techniques is the link between individual protein variants and their corresponding genetic information. To provide this link, the most successful combinatorial protein engineering methods employ microorganisms, such as bacteriophages, bacteria or yeast for the production and display of libraries. This thesis focuses on the development and application of new directed evolution methods utilizing the bacterium Escherichia coli (E. coli), for the engineering of affinity proteins and proteases.

The first study aimed to engineer the substrate specificity of a protease from tobacco etch virus (TEV). For this purpose, a novel method was devised based on expression of intracellular protease libraries, and employed a reporter fusion protein consisting of amyloid β peptide fused to the N-terminus of enhanced green fluorescent protein (EGFP). Variants were screened for proteolytic activity on co-expressed target substrate by means of fluorescence-activated cell sorting (FACS). After three rounds of FACS, a set of TEV protease variants were enriched that exhibited improved proteolytic activity on the novel substrate.

Studies two to four describe the development of an E. coli surface expression system that was explored for directed evolution applications. The method is based on display of recombinant proteins on the outer membrane via fusion to a bacterial autotransporter, adhesin involved in diffuse adherence I (AIDA-I). The second study focused on the optimization of the surface display system and its application to directed evolution. In this effort, several affinity protein classes were evaluated for surface display via AIDA-I in a panel of E. coli strains. Results showed that smaller and less complicated affibody molecules were displayed at high levels, while more complex proteins, such as antibody fragments, varied in their performance and functioned best in certain engineered strains. A mock affibody library was used to develop a high-throughput magnetic-assisted cell sorting (MACS) protocol for enrichment of binders from very large libraries.

In the third and fourth study, the new E. coli display method combined with the MACS protocol was evaluated for generation of new affibody molecules.

In the third study, a large naïve affibody library (>1.5×1011 members) was constructed, displayed on E. coli and characterized. The performance of the method and library was evaluated by selection of binders against two cancer-associated targets, tumor-associated calcium signal transducer 2 (TROP-2) and lymphocyte-activation gene 3 (LAG-3). MACS and FACS were performed, with flow cytometry assessment between rounds to monitor enrichment. Both selections produced high affinity binders to their respective targets.

In the fourth study, a maturation library was constructed for improving the properties of an affibody molecule toward the renal cell carcinoma biomarker carbonic anhydrase IX (CAIX). Selections included stringent off-rate procedures and yielded variants with improved affinities and folding stability compared to previously reported binders.

In summary, the work in this thesis demonstrate the potential of E. coli-based directed evolution methods for selection of new proteins with altered or improved properties.