PUREfrex Reaction Supplements

state-of-the-art protein synthesis by

PUREfrex® supplements expand the potential of base kits.

yyyy Molecular Chaperone Supplements Promote Protein Solubility and Protein Folding
Molecular chaperones such as hsp70 (e.g. E. coli DnaK) assist the correct folding of the newly synthesized proteins. Because base PUREfrex® kits do not contain molecular chaperones, some proteins may have difficulty forming the correct conformation and be insoluble after synthesis. In such cases, molecular chaperones could contribute to make them soluble and active.

Chaperone Supplement

DnaK Mix comprises highly purified and ratio-optimized E. coli DnaK, DnaJ and GrpE. DnaK, known as Hsp70, has ATPase activity and is stimulated by co-chaperones DnaJ and GrpE. DnaJ facilitates DnaK ATPase activity and binds to hydrophobic regions of synthesized proteins. GrpE stimulates ADP/ATP exchange of DnaK. DnaK Mix works well with PUREfrex^®series and DsbC Set.

Reagent	Qty	Description	Storage
DnaK Mix	25 ul	100µM DnaK, 20µM DnaJ and 20µM GrpE from E.coli in 30% glycerol buffer (No tags)	-80 C
Dilution Buffer	500 ul	30% glycerol buffer	-20 C

Chaperone Supplement

GroE Mix comprises highly purified and ratio-optimized E. coli GroEL (known as Hsp60) and GroES (works in conjunction with GroEL). 

  

Reagent	Qty	Description	Storage
GroE Mix	12.5 ul	20µM GroEL, 14-mer form, and 40µM GroES, 7-mer form, from E.coli in 30% glycerol buffer (No tags)	-80 C
Dilution Buffer	500 ul	30% glycerol buffer	-20 C

 

Redox Environment Supplements Promote Disulfide Bond Formation
Formation of disulfide bonds is important for folding and stability of secretory proteins such as enzymes or antibodies. Disulfide bonds are usually formed by oxidation of sulfhydryl groups (SH-) of adjacent cysteine residues. Therefore, disulfide bond formation efficiency depends on redox state. Additionally, disulfide bond isomerase which can catalyze the exchange of disulfide bridges may be required for correct cysteine pairing.

Redox Supplement

DsbC Set comprises highly purified E. coli DsbC (a disulfide bond isomerase) which can catalyze disulfide bridge exchange, and GSSG (oxidized Glutathione) to enforce an oxidized environment.

 

• Reagent	Qty	Description	Storage
  GSSG	25 ul	Oxidized glutathione (60 mM)	-20 C
  DSBC	25 ul	E.coli DsbC (320 µM *2) (No tags)	-80 C
  Dilution Buffer	500ul	30% glycerol buffer	-20 C

Redox Supplement

PDI Set comprises oxidized glutathione (GSSG), human PDI (protein disulfide isomerase) and human Ero1α (ER oxidoreductin-1 to reoxidize PDI).

• Reagent	Qty	Description	Storage
  GSSG	25 ul	Oxidized glutathione (60 mM)	-20 C
  PDI	25 ul	Human protein disulfide isomerase with no tags (200 µM)	-80 C
  Ero1a	25 ul	Human ER oxidoreductin-1 to reoxidize PDI with no tags (5 µM)	-80 C
  Dilution Buffer	500 ul	30% glycerol buffer	-20 C

 

Elongation Factor P (EF-P) promotes translation of consecutive proline residues.
The E. coli translation factor EF-P (elongation factor P), a bacterial homolog of eukaryotic initiation factor 5A (eIF5A), functions to promote peptide bond formation between the first methionine and the next amino acid as well as the elongation of consecutive proline residues (3 or more residues). In bacteria, LYS34 of EF-P is modified post translationally to ß-lysllysine by EpmA (YjeA/GenX) and EpmB (YjeK). This modification is important to EF-P activity. PUREfrex EF-P Supplement contains this modification at LYS34.

Transcription Factor Supplement

EF-P Supplement comprises recombinant EF-P (modified at LYS34) and dilution buffer. Standard PUREfrex® kits do not contain EF-P. Addition of EF-P to PUREfrex reactions can improve the yield of proteins containing consecutive proline residues such as Pro-Pro-Pro and Pro-Pro-Gly by preventing ribosome stalling and promoting the formation of peptide bonds.

 

Reagent	Qty	Description	Storage
EF-P	12.5 ul	Elongation Factor P (40µM) (No tags)	-80 C
Dilution Buffer	500 ul	30% glycerol buffer	-20 C

 

PUREfrex Ordering Information
Product Name (click for price and order info)	Catalog Number	Total Reaction Volume	Notes	Product Information Page
Kits
PUREfrex 2.0	GFK-PF201-0.25-EX	250 ul		2.0
	GFK-PF201-0.25-5-EX	5 x 250 ul (packaged as 5 x 250 ul)	Sales to be discontinued
	GFK-PF201-2ML-EX	1 x 2 ml	New size !
	GFK-PF201-10ML-EX	5 x 2 ml	Change in catalog number
Current generation kit for protein synthesis. Contains DTT as a reducing agent.
PUREfrex 2.1	GFK-PF213-0.25-EX	250 ul		2.1
	GFK-PF213-0.25-5-EX	5 x 250 ul (packaged as 5 x 250 ul)	Sales to be discontinued
	GFK-PF213-2ML-EX	1 x 2 ml	New size !
	GFK-PF213-10ML-EX	5 x 2 ml	Change in catalog number
Current generation kit for proteins with disulfide bonds. Formulated for user control of redox environment.
PUREfrex 1.0	GFK-PF001-0.25-EX	250 ul		1.0
	GFK-PF001-0.25-5-EX	1250 ul (packaged as 5 x 250 ul)	Sales to be discontinued
	GFK-PF001-2ML-EX	1 x 2 ml	New size !
	GFK-PF001-10ML-EX	5 x 2 ml	Change in catalog number
Prior generation kit for protein science research and synthetic biology. Components are fully disclosed. Custom formulated kits are based on 1.0 components. Please inquire about custom kits.
PUREfrex Reaction Supplements
DnaK Mix	GFK-PF003-0.5-EX	for 500 ul of reaction mix		Supplements
	GFK-PF003-10ML-EX	5 x 2 ml	Change in catalog number
GroE Mix	GFK-PF004-0.5-EX	for 500 ul of reaction mix		Supplements
	PF004-10ML-EX	5 x 2 ml	Change in catalog number
Supplements for synthesis of aggregate-prone proteins.
DsbC Set	GFK-PF005-0.5-EX	for 500 ul of reaction mix		Supplements
	PF005-10ML-EX	5 x 2 ml	Change in catalog number
PDI Set	GFK-PF006-0.5-EX	for 500 ul of reaction mix		Supplements
	PF006-10ML-EX	5 x 2 ml	Change in catalog number
Supplements for synthesis of proteins containing disufide bonds.
EF-P	GFK-PFS052-0.5-EX	for 500 ul of reaction mix		Supplements
	PFS052-10ML-EX	5 x 2 ml	Change in catalog number
Supplement for use when consecutive prolines are in target protein.