The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology 
The Hebrew University of Jerusalem
Dr. Mario Lebendiker
mario.l@mail.huji.ac.il  Tel: 972-2-6586920 

Additives Used to Stabilize Folding and Prevent Aggregation

Summary of different articles:

        De Bernardez Clark et al.,  (1999)  Inhibition of aggregation side reactions during in vitro protein folding. METHODS IN ENZYMOLOGY, VOL. 309: 217–236

        Voziyan P. et al. (2000). Refolding a glutamine synthetase truncation mutant in vitro: Identifying superior conditions using a combination of chaperonins and osmolytes.                                     Journal of Pharmaceutical Science 89: 1036

        Goloubinoff et al. (2001) Chemical Chaperones Regulate Molecular Chaperones in Vitro and in Cells under Combined Salt and Heat Stresses. J. Biol. Chem., Vol. 276, Issue 43, 39586-39591

        Bondos S. and Bicknell A. (2003) Detection and prevention of protein aggregation before, during, and after purification.  Analytical Biochemistry 316: 223-231

        Golovanov, A. et.al (2004)  A simple method for improving protein solubility and long-term stability.     J. Am. Chem. Soc. 126: 8933–8939

        Hamada, H. et al. (2009) Effect of Additives on Protein Aggregation. Current Pharmaceutical Biotechnology, 10: 400-407

        Shukla, D. et al., (2011), Molecular level insight into intra-solvent interaction effects on protein stability and aggregation.  Adv. Drug Deliv. Rev. doi:10.1016/j.addr.2011.06.014


        Churion, K. & Bondos, S. (2012) Identifying solubility-promoting buffers for intrinsically disordered proteins prior to purification. Methods in Molecular Biology  896: 415-427

        Leibly, D.J. et al., (2012) Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins. Plos One 7(12): e52482

        DILYX Biotechnologies   OptiSol Protein Solubility Screening  Kit  Application Manual

       HAMPTON:  Solubility & Stability Screen is designed to assist in the identification of solution conditions which promote protein solubility and stability,
            and minimize protein precipitation. Solubility & Stability Screen is a solubility screen, a stability screen, and may also be used as an additive screen
            in the presence of a crystallization reagent.   (pdf-I)   (pdf-II)

 

 Additive

Recommended Initial Concentration

Recommended Concentration Range

 

Sugars and Osmolytes

glycerol

10%

0-40%

TMAO  (trimethylamine N-oxide) 

0.5M

0-1 M

Glucose

0.5M

0-2 M

Sucrose

0.5M

0-1 M

Trehalose

0.5M

0-1 M

Ethylene glycol

10%

0-60%

D-Sorbitol

0.2-1M 

0.2-1M

Mannitol

Mannitol 5%- Glycine 5%

2%

5% - 5%

 

Xylitol

0.2-1M

0.2-1M

Glycine Betaine

1M

 

 

 

 

Amino acids and Amino acid derivatives

Glycine

Mannitol 5%- Glycine 5%

50-250mM 

5% - 5%

0.5-2%

Arginine L-HCl

25-125mM

0-2 M

Arginine Ethylester

50-250mM 

0-500 mM

Proline

50-250mM

0-1 M

Potassium Glutamate

 

0-500 mM

Arginine L-HCl +  L-glutamic acid (L-Glu)

50mM each

 

 

 

 

Non-ionic Detergents

Nonidet P40 (NP40) or Triton X-100

0.01%

0-1%

Tween 80  or 20

0.1%

0-1%

DDM: n-Dodecyl β-D-maltoside

 

0.01- 0.5%

Brij 56 (Polyoxyethylene cetyl ether

0.05%

 

OG: Octyl glucoside (n-octyl-β-D-glucoside)

 

0.01- 0.5%

Poloxamer 188 (BASF Pluronic® F68)

 

 

 

 

 

Zwiterionic Detergents

NDSB: Non-detergent Sulfo Betaine

0.5M

0-1M

CHAPS

 

0.01- 0.5%

Zwittergent 3-14

 

0.001-0.2 %

LDAO:  Lauryldimethylamine  N-oxide

 

0.01- 0.5%

 

 

 

Ionic Detergents

CTAB: cetyltrimethylammonium bromide

0.5%

 

Sarkosyl : Sodium lauroyl sarcosinate

0.05%

0.01-0.5%

SDS

 

Up to 0.1%

 

 

 

Mild chaotropes agents and chaotropes salts

Urea

0.5M

0-2M

Guanidine HCl

0.5M

0-2M

N-Methylurea

 50-250mM

up to 2.5M

N-Ethylurea 

20-100mM 

up to 2M
 N-Methylformamide 3-15%

NaI

0.2M

0-0.4 M

CaCl2

10-50 mM

0-0.2M

MgCl2

10-50 mM

0-0.2M

 

 

 

 

 

 

Mild and strong kosmotropes salts

NaCl (weak)

300mM

0-1 M

KCl (weak)

200mM

0-1 M

MgSO4 (strong)

100mM

0-0.4 M

(NH4)2SO4 (strong)

50mM

0-0.2M

Na2SO4 (strong)

500mM

0-0.2M

Cs2SO4 (strong)

50mM

0-0.2M

Potassium citrate

100mM

 

Citric Acid

50mM

 

 

 

 

Alcohols, Polyols, Polymer, Polyamines and others

Ethanol

5-10%

Up to 25%

n-Penthanol

 

1 to 10mM

n-Hexanol

 

0.1 to 10mM

Cyclohexanol

 

0.01 to 10mM

Polyethylene glycol (PEG 3350)

0.3-1.5% 

0.1-0.4 g/L

Polyvinylpyrrolidone  40 (PVP40)

 

 0.05-4%

Alpha-Cyclodextrin

8-40mM

 

Beta-cyclodextrin

1-5mM

 
 Putrescine, spermidine, and  spermine 0.1M

Formamide

0.1%

 

 

 
HOME
EXPRESSION SYSTEMS
EXTRACTION AND CLARIFICATION
PURIFICATION
CHARACTERIZATION
OTHERS


Dr. Mario Lebendiker The Protein Purification Facility
The Wolfson Centre for Applied Structural Biology,    The Hebrew University of Jerusalem
mario.l@mail.huji.ac.il  Tel: 972-2-6586920  FAX: 972-2-6758963

Copyright ©, 2002, The Hebrew University of Jerusalem. All Rights Reserved.