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Recombinant protein production is pivotal in molecular biology, enablingprofound insights into cellular processes through biophysical, biochemical, andstructural analyses of the purified samples. The demand for substantialbiomolecule quantities often presents challenges, particularly for eukaryoticproteins. Escherichia coli expression systems have evolved to address theseissues, offering advanced features such as solubility tags, posttranslationalmodification capabilities, and modular plasmid libraries. Nevertheless,existing tools are often complex, which limits their accessibility andnecessitates streamlined systems for rapid screening under standardizedconditions. Based on the Golden Gate cloning method, we have developed a simple'one-pot' approach for the generation of expression constructs usingstrategically chosen tags like hexahistidine, SUMO, MBP, GST, and GB1 toenhance solubility and expression. Tags are removable via TEV proteasecleavage, and the system allows visual cloning verification through mScarletfluorescence. We provide a comprehensive protocol encompassing oligonucleotidedesign, cloning, expression, Ni-NTA affinity chromatography, and size-exclusionchromatography. This method therefore streamlines prokaryotic and eukaryoticprotein production, rendering it accessible to standard molecular biologylaboratories with basic protein biochemical equipment.

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