SURF 2024: Making Purer PURE

2024 SURF project description

• Mentor: Richard Murray
• Co-mentors: Yan Zhang and Zachary Martinez

Main Objective: Reduce background contaminant of One-Pot PURE systems

Introduction

The PURE (Protein Synthesis Using Recombinant Enzymes) system uses a defined set of 36 enzymes necessary for transcription, translation, and energy cycling to create a minimal system to support protein expression. This minimal system allows facile removal or replacements of specific enzymes involved in reactions with the potential to power impactful applications in prototyping novel protein designs via complementation assays and producing therapeutic proteins via codon replacement for non-canonical amino acids.

Despite the promise of PURE, current homemade One-Pot PURE systems relying on metal-affinity-based purification exhibit high background containments in the purified product. Although traditional single protein purification methods can be interfaced with downstream chromatography steps to achieve high product purity, such processing steps are incompatible with the purification of One-Pot PURE, which involves the simultaneous purification of 36 proteins spanning 7 – 110 kDa. To equip researchers with the capability to develop truly defined and pure PURE systems, a new method to create a purer PURE system is necessary.

Motivation

Current homemade and One-Pot PURE systems rely on traditional 6xHis tags for protein purification. This results in high background contaminants in the purified product – particularly metal-binding proteins. To enable impactful biological applications where the absence of background contaminants is critical, we need an approach to developing the purer PURE system. The central hypothesis of this project is to investigate the use of peptide- and protein-based tags to produce a PURE system without background contamination.

Project Overview

This project investigates whether peptide- and protein-based purification tags can lead to a purer PURE system. The key milestones of the projects are delineated as follows:

1. To prevent the purification of truncated products, this project is limited to identifying suitable affinity tags for C-terminal incorporation.

2. Selecting optimal purification tag candidates that minimize disruption to protein folding and enzymatic activity using predictive protein structure models.

3. Cloning screened purification tag candidates for 36 enzymes constituting the PURE system.

4. Verifying that the One-Pot PURE system made with new affinity tags has significantly less background protein contamination than the traditional his-tagged method using mass spectrometry-based proteomics.

5. Characterizing the protein yield and reaction kinetics of traditional and “purer” One-Pot PURE systems.

Preferred Skills

Background in basic molecular biology through coursework

Some experimental experience through coursework with a lab component or research

Students with structural biology background are highly preferred

References

Lavickova, B. and Maerkl, S. J. (2019). A simple, robust, and low-cost method to produce the pure cell-free system. ACS Synthetic Biology, 8(2), 455-462. https://doi.org/10.1021/acssynbio.8b00427