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 Ni-His 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 the concentration of each protein by

Pierce660 Assay.

Calculate how much volume of each protein stock is needed for a given final volume of Protein Mix (here: 100 uL)

#	Protein	Protein Mix Concentration [ng/uL]	Stock Concentration [ng/uL]	Volume to Add per 100 uL Protein Mix [uL]
1	AlaRS	538
2	ArgRS	15
3	AsnRS	169
4	AspRS	62
5	CysRS	9
6	GlnRS	29
7	GluRS	97
8	GlyRS	74
9	HisRS	6
10	IleRS	308
11	LeuRS	31
12	LysRS	49
13	MetRS	18
14	PheRS	131
15	ProRS	77
16	SerRS	15
17	ThrRS	48
18	TrpRS	48
19	TyrRS	5
20	ValRS	14
21	IF1	77
22	IF2	308
23	IF3	77
24	EF-G	385
25	EF-Tu	3846
26	EF-Ts	385
27	RF1	77
28	RF2	77
29	RF3	77
30	RRF	77
31	MTF	154
32	CK	31
33	MK	23
34	NDK	8
35	PPiase	8
36	T7RNAP	77
	Total	7430

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

Aliquot Protein Mix (e.g., eight (8) PCR tubes of 12.5 uL each) and store at -80C until ready for use.

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