Simplifying recombinant protein production: Combining Golden Gate cloning with a standardized protein purification scheme

TL;DR

This paper introduces a streamlined, standardized method combining Golden Gate cloning with a universal protein purification scheme to simplify recombinant protein production, making it more accessible and efficient for various laboratories.

Contribution

It presents a novel one-pot cloning approach with a standardized purification protocol, integrating multiple tags and visual verification for rapid protein expression screening.

Findings

Efficient generation of expression constructs using Golden Gate cloning.

Versatile tagging system for enhanced solubility and expression.

Simplified protocol suitable for diverse protein production needs.

Abstract

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitates streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple 'one-pot' approach for the generation of expression constructs using strategically chosen tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. Tags are removable via TEV protease cleavage, and the system allows visual cloning verification through mScarlet fluorescence. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, Ni-NTA affinity chromatography, and size-exclusion chromatography. This method therefore streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.