Protein Mix (PMix)

Overview¶

Protein Mix (PMix) contains the 36 proteins of the PURE system at a total of 15 µg/µL in Protein Buffer: the 20 aminoacyl-tRNA synthetases, three initiation factors, three elongation factors, four release factors, five metabolic enzymes (MTF, CK, AK, NDK, PPiase), and T7 RNA polymerase (T7RNAP). With Small Molecule Mix (SMix), ribosomes, and tRNA, it makes Base Cytosol.

Reference Composition¶

Proteins

Small Molecules

Composition of the Protein Mix (PMix) at stock concentration. Molecular weight, stoichiometry, and molarity are computed as described in the note below.

Category	Protein	Concentration (ng/µL)	Molecular Weight (kDa)	Stoichiometry	Molarity (µM)
tRNA Synthetases	AlaRS	1086	96.0	Homotetramer	2.83
	ArgRS	30	64.7	Monomer	0.464
	AsnRS	341	52.6	Homodimer	3.24
	AspRS	125	65.9	Homodimer	0.948
	CysRS	18	52.2	Monomer	0.345
	GlnRS	59	63.5	Monomer	0.929
	GluRS	196	53.8	Monomer	3.64
	GlyRS*	149	223.2	Heterotetramer (α₂β₂)	0.668
	HisRS	12	47.0	Homodimer	0.128
	IleRS	622	104.4	Monomer	5.96
	LeuRS	63	97.2	Monomer	0.648
	LysRS	99	57.6	Homodimer	0.859
	MetRS	36	76.3	Homodimer	0.236
	PheRS*	264	248.4	Heterotetramer (α₂β₂)	1.06
	ProRS	155	63.7	Homodimer	1.22
	SerRS	30	48.4	Homodimer	0.310
	ThrRS	97	74.0	Homodimer	0.655
	TrpRS	97	37.4	Homodimer	1.30
	TyrRS	10	47.5	Homodimer	0.105
	ValRS	28	108.2	Monomer	0.259
Initiation Factors	IF1	155	8.2	Monomer	18.9
	IF2	622	97.3	Monomer	6.39
	IF3	155	20.6	Monomer	7.52
Elongation Factors	EF-G	777	77.6	Monomer	10.0
	EF-Tu	7764	43.3	Monomer	179
	EF-Ts	777	30.4	Monomer	25.6
Release Factors	RF1	155	40.5	Monomer	3.83
	RF2	155	41.3	Monomer	3.75
	RF3	155	59.6	Monomer	2.60
	RRF	155	20.6	Monomer	7.52
Metabolism	MTF	311	34.2	Monomer	9.09
	CK	63	43.3	Homodimer	0.727
	AK	46	23.6	Monomer	1.95
	NDK	16	15.5	Homohexamer	0.172
	PPiase	16	19.7	Homohexamer	0.135
Transcription	T7RNAP	155	98.9	Monomer	1.57
Total		15 000

Small molecules of the Protein Mix (PMix) at stock concentration. All come from Protein Buffer with 30 % glycerol.

Component	Concentration
HEPES-KOH (pH 7.6)	50 mM
Potassium chloride	100 mM
Magnesium chloride	10 mM
TCEP	1 mM
Glycerol	300 mL/L

Processes¶

Make Protein Mix — from the 36 proteins, each purified on its own
Make OnePot Protein Mix — makes OnePot Protein Mix, a functional equivalent of PMix with the same proteins at molar ratios that cannot be controlled
Assemble Base Cytosol — 1.2 µL of PMix per 10 µL reaction

Credits¶

Reformulated by Yemo Ku and Jon Calles (b.next) from the PURE system, developed by the Ueda Lab (Shimizu et al., 2001).

References¶

Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), 751–755. 10.1038/90802