PURE (”Protein Synthesis Using Recombinant Elements”) is a defined set of molecules (proteins, RNAs, small organic molecules, and salts) formulated in 2001 by Shimizu and colleagues to recapitulate transcription and translation. PURE is widely used in synthetic cell engineering.
PURE is broadly composed of two (2) classes of components:
- Protein Mix: thirty-six (36) proteins plus ribosomes that reconstitute transcription, translation, amino acylation, and energy recycling.
- Energy Mix: thirty-one (31) small organic molecules and salts, plus tRNAs, that support transcription and translation.
PURE is available commercially under the product names PUREfrex (GeneFrontier Corporation) and PURExpress (NEB) in small quantities (~ 1 mL). PURE can also be produced by sourcing the individual components and assembling Protein Mix and Energy Mix. Of these, Protein Mix is far harder to make, requiring many laborious protein purification steps.
Several published protocols aim to make Protein Mix production more accessible, primarily by co-culturing expression strains and co-purifying proteins (e.g., TraMOS PURE and OnePot PURE). These pooled purification methods are much cheaper and more convenient, but are surprisingly tricky to get working the first time, and to get working reproducibly (across batches and across labs). We recommend new users first make batches of Protein Mix by individually purifying each protein before attempting co-culture or co-purification.
Make Protein Mix - Prepare the consumables you will need. Make buffers and cell culture media. Assemble gravity columns, pack them with affinity resin, equilibrate, and store.
Prepare Columns, 
Make Protein Purification Buffers and Media - Grow out E.coli expression strains expressing the thirty-six (36) protein of PURE, induce protein expression, and collect bacterial pellets for downstream purification.
Grow and Induce Expression Strains - Lyse your bacteria using a sonicator, allowing you to access the proteins inside. The resulting mixture of cell debris and target proteins is called “lysate”. Remove debris by centrifugation and filtering to make “clarified lysate”.
Lyse Bacteria by Sonication - Purify your target proteins from clarified lysate by flowing them through your affinity columns. PURE proteins are expressed with a polyhistidine affinity tag, which selectively binds Ni2+ affinity resin. Flow your clarified lysate through your columns, wash out debris, and elute your target proteins with a high salt (Imidazole) buffer.
Purify Proteins by Ni2+ Gravity Column - Wash and Concentrate your proteins simultaneously using a centrifugal filter. Load your sample, remove the high salt buffer by centrifugation. Then, wash with fresh buffer, centrifuge, and repeat. Finally, centrifuge to remove buffer from your sample until you reach your target concentration.
Exchange Buffers and Concentrate by Spin Filtration - Measure the concentration and purity of your proteins by protein gel electrophoresis and Pierce660 Assay, respectively.
Protein Gel 
Pierce660 Assay. - Assemble Protein Mix by combining each purified protein at its specified concentration. Aliquot and store at -80C. Assemble Protein Mix
Make Energy Mix - Make tRNAs by growing out an E. coli culture, extracting small nucleic acids with acid phenol, and purifying tRNAs from small DNA by alcohol precipitation. Measure the purity and concentration of your tRNAs by urea gel elecrophoresis and UV-Vis spectroscopy (Nanodrop), respectively.
Make tRNAs
- Make Amino Acid Mix by weighing, resuspending, and combining twenty (20) amino acids. Make Amino Acid Mix
- Assemble Energy Mix. Prepare the remaining eleven (11) components of Energy Mix. Then, combine with tRNA and Amino Acid Mix. Aliquot and store at -80C. Make Energy Mix . Make Energy Mix
Assemble PURE Reactions - Make Ribosomes by growing out an E. coli culture, purify ribosomes by FPLC and ultracentrifugation. Measure the purity and concentration of your ribosomes by protein gel electrophoresis and UV-Vis spectroscopy (Nanodrop), respectively. Aliquot and store at -80C.
Make Ribosomes
- Assemble PURE reactions.