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 laborious protein purification steps, 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 Columns and 
Make Protein Purification Buffers and Media - get ready to purify proteins by making all the buffers and media you will need, as well as prepare the columns that you will use to purify the proteins.
Grow and Induce Expression Strains - Using thirty-six (36) E. coli expression strains, grow bacterial cultures, induce protein expression, and collect bacterial pellets for downstream purification.
Lyse Bacteria by Sonication - Take your pellets and break open the bacteria using a sonicator so that you can access the proteins inside. The resulting mixture of cell debris and target proteins is called “lysate”.
Purify Proteins by Ni-His Gravity Column - Purify your target proteins from lysate by running them over an affinity column. PURE proteins have a purification tag (6xHis) that makes them selectively bind the column, allowing the cell debris to wash away. Elute PURE proteins off of the column using a high salt (Imidazole) buffer.
Exchange Buffers and Concentrate by Spin Filtration - Take your purified proteins out of elution buffer and concentrate them down to easier to handle concentrations by using centrifugal filters.- Check the purity of your protein preps by
Protein Gel. Check the concentration of your protein stocks by 
Pierce660 Assay - Assemble Protein Mix - Assemble proteins into Protein Mix.
Make Energy Mix - Assemble Amino Acid Mix - One buffer stock for each of twenty (20) amino acids.
- Make twelve (12) other component buffers (see Make Energy Mix)
- Combine Amino Acid Mix and component buffers into Energy Mix (see Make Energy Mix)
Assemble PURE Reactions