Assemble Protein Mix

Getting Started

You want to make PURE. You’ve got the thirty-six (36) purified proteins that make up the protein component of PURE and you want to assemble Protein Mix from them. This protocol tells you how to assemble Protein Mix from individually purified proteins.

Prerequisite Protocols

Purify PURE proteins:

Grow and Induce Expression Strains,

Lyse Bacteria by Sonication,

Purify Proteins by Ni2+ Gravity Column,

Exchange Buffers and Concentrate by Spin Filtration

Measure the concentration of your proteins:

Pierce660 Assay

Protocol

Thaw your protein stocks on ice.

Measure your protein stock concentrations by

Pierce660 Assay.

Read the following Google Sheet template and follow its directions to assemble your Protein Mix. https://docs.google.com/spreadsheets/d/1OLFNVuiL5-f6FAD-eY3rEHRrYya3oPlh2JK1UhwY7qs/edit?usp=sharing

Untitled

Input the following information into blue colored cells:

Desired number of 10 uL PURE reactions

Protein stock concentrations

Protein stock volumes

Check the “Checklist” section to see if any cells are highlighted yellow. If so, follow the directions next to the highlighted cell.

‣

Notes: if your Protein Mix is dilute, you can concentrate it

Concentrate Protein Mix to the Target Final Volume (~ 12.25 mg / mL) using a 3kDa Amicon Centrifugal Filter.

‣

Notes: wash and concentrate your Protein Mix after assembly

Measure the total protein concentration of your Protein Mix:

Create 10x dilutions of Protein Mix (1 uL in 9 uL Ultrapure water) in triplicate.

Perform a

Pierce660 Assay on your diluted Protein Mix samples.

Calculate the total protein concentration of your diluted Protein Mix samples. Average the values of each triplicate and multiply by 10 to estimate the total protein concentration of your Protein Mix.

Dilute your Protein Mix to 12.25 mg / mL in P3 Storage Buffer (30% glycerol). If the concentration of your Protein Mix is less than 12.25 mg / mL, repeat the last two steps (concentrate your mix, then measure using Pierce660)

Aliquot your Protein Mix (12 uL + 1 uL of head room per tube) into PCR tubes or microcentrifuge tubes and store -80C.

Resources and References

Papers

Original PURE paper

Cell-free translation reconstituted with purified components

Nature Biotechnology - Cell-free translation reconstituted with purified components

doi.org

Cell-free translation reconstituted with purified components

OnePot PURE

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

We demonstrate a simple, robust, and low-cost method for producing the PURE cell-free transcription–translation system. Our OnePot PURE system achieved a protein synthesis yield of 156 μg/mL at a cost of 0.09 USD/μL, leading to a 14-fold improvement in cost normalized protein synthesis yield over existing PURE systems. The one-pot method makes the PURE system easy to generate and allows it to be readily optimized and modified.

doi.org

A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

OnePot PURE Cell-Free System | Text Page

Scientific Article | The defined PURE (protein synthesis using recombinant elements) transcription-translation system provides an appealing chassis for...

dx.doi.org

OnePot PURE Cell-Free System | Text Page

Credits

Yan Zhang, Zoila Jurado, and Miki Yun (Richard Murray Lab, Caltech)

Developers