  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.
