Biofoundry-assisted expression and characterization of plant proteins

Abstract

Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterization has remained a technical bottleneck, often requiring significant effort to optimize expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichia coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification and improved expression/folding. We next optimized automated assembly of miniaturized cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11-amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.

Keywords: automation; biofoundry; cell-free protein synthesis; golden gate DNA assembly; plant biotechnology.

Figure 1.

A workflow for biofoundry-assisted DNA assembly and cell-free protein synthesis (CFPS) of plant proteins. (A) Level 0 DNA parts (phytobricks) encoding the protein of interest can be assembled into functional expression plasmids for CFPS or expression in planta. Acoustic liquid handling enables the screening of libraries of protein variants to determine optimal expression configurations using the HiBiTLgBiT luminescence. (B) The CFPS cloning toolbox consists of acceptor plasmids that assemble with a CDS and C-terminal tag along with an optional N-terminal tag. The cloning overhangs are compatible with existing plant DNA assembly standards.

Figure 2.

(A) Cell-free expression of sfGFP fused to a variety of N- and C-terminal tags. CFPS reactions were run at the 15-μl scale and incubated for 20 h at 30°C. Relative protein expression was measured by GFP fluorescence and normalized to pEPQDKN0248 quantified by HiBiT. (B) Removal of N-terminal tags by mixing a CFPS reaction expressing TEV protease (1:130 w/w ratio of TEV protease to substrate protein) and incubating at 16 h at 30°C. Protein visualized by western blot with an α-GFP-HRP antibody. (C) Expression of GFP variants from panel A with reactions assembled manually using handheld pipettes (15-μl reactions) or automated using an Echo 550 acoustic liquid handler (2-μl reactions). Values in panels A and C represent averages (n = 3), and error bars represent 1 SD.

Figure 3.

(A–B) HiBiT quantification of plant proteins with various N-terminal tags with cell-free protein synthesis reactions assembled using acoustic liquid handling platforms. (A) Expression of 11 plant UGTs, each with eight different N-terminal tags. (B) Expression of five plant transcription factors, each with five different N-terminal tags. (C) Comparison of E. coli CFPS (with NET tag) and the TNT® SP6 High-Yield Wheat Germ Protein Expression System (Promega) for expressing wheat transcription factors at the 15-μl scale. Values represent averages (n = 3), and error bars represent 1 SD.

Figure 4.

Plant proteins expressing using E. coli cell-free protein synthesis are functionally active. (A) UDPglycosyltransferase UGT73C5 from Arabidopsis thaliana can glycosylate geraniol or nepetalactol when purified (blue trace) or when expressed using CFPS (red, orange, teal trace) and (B) chrysanthemol diphosphate synthase (CcCPPase) from Chrysanthemum cinerariaefolium converts substrate DMAPP to chrysanthemol and lavandulol. (C) Cell-free-expressed AtTGA2 binds a 60-bp DNA probe containing its cognate-binding site but does not impede the mobility of a probe with randomized sequence.