Ectoine / Hydroxyectoine - ARTIMMUN · Hydroxyectoin: C6H10N2O3, Mr 158.2, solubility in water: 4.2 M. Bulk quantities of Ectoine and Hydroxyectoine are available on request. Introduction

bitop AG I Stockumer Straße 28 I 58453 Witten I Germany I Phone: +49 (0) 2302-91 440-0 I [email protected] I www.bitop.debitop AG I Stockumer Straße 28 I 58453 Witten I Germany I Phone: +49 (0) 2302-91 440-0 I [email protected] I www.bitop.de

Ectoine / Hydroxyectoine

Order Number Description Order Number Description

LC101-0001 LC101-0010 LC101-0100

Ectoine ultra pure 99%, 1 g Ectoine ultra pure 99%, 10 g Ectoine ultra pure 99%, 100 g

LC103-0001 LC103-0010 LC103-0100

Hydroxyectoine ultra pure 99%, 1 g Hydroxyectoine ultra pure 99%, 10 g Hydroxyectoine ultra pure 99%, 100g

LC102-0001 LC102-0010 LC102-0100

Ectoine pure 95%, 1 g Ectoine pure 95%, 10 g Ectoine pure 95%, 100 g

LC104-0001 LC104-0010 LC104-0100

Hydroxyectoine pure 95%, 1 g Hydroxyectoine pure 95%, 10 g Hydroxyectoine pure 95%, 100 g

Ectoin: C6H10N2O2, Mr 142.2, solubility in water: 4.0 M. Hydroxyectoin: C6H10N2O3, Mr 158.2, solubility in water: 4.2 M. Bulk quantities of Ectoine and Hydroxyectoine are available on request.

Introduction

Ectoine and Hydroxyectoine are Extremolytes isolated from the halophilic bacterium Halomonas elongata. The Ectoines are the best characterized Extremolytes and their ability to stabilize a variety of proteins, nucleic acids, membranes and whole cells is well documented. The Ectoines are highly water-soluble, zwitterionic low-molecular weight molecules. They are biologically inert, they do not interfere with most enzymatic and binding reac-tions and are highly compatible with cell metabolism and tolerated in their producer organisms up to concentrations of 1 M. Recommended Usage

Ectoines can be used in a wide range of applications for the protection and stabilization of biological macromolecules and cells. The Ectoines are capable to protect and stabi-lize proteins in vitro and in vivo. Possible applications are stabilization of diluted protein solutions, stabilization during freeze-drying, freeze-thaw cycles and heat stabilization. Product safety and handling

This product is considered non-hazardous as defined by the Hazard Communication Standard. Avoid contact with skin and eyes. In case of contact or spillage, clean with copious amounts of water. Storage, Shipment and Stability

Store at 2…30°C. This product is DNAse-, RNAse and Protease-free and contains no proteins and preservatives. Ectoine and Hydroxyectoine have a shelf life of 12 month. This product is intended for in-vitro use only. Please contact bitop AG or your distributor for more details.

Distributed by Artimmun Analytik GmbH [email protected], T 49-6195-674724, F--674722

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Examples: Ectoines can stabilize the complete range of bio molecules. Some examples of tested mole-cules and stress situations are listed below.

Protein Stress Situation Protein conc.

Final Concentration Residual

Activity (%) Lactate Dehydrogenase 52 µg/ml 1.0 M Hydroxyectoine 100 Phosphofructokinase 75 µg/ml 1.0 M Ectoine 100 Enolase 50 µg/ml 0.4 M Ectoine 100 Glutamate Dehydro-genase

350 µg/ml 0.5 M Hydroxyectoine 85

Carboxylesterase 200 µg/ml 0.5 M Hydroxyectoine 100 CD30-Binding-protein

fast freeze/ slow thaw (4x) dT = -196°C in 30 sec. dT = + 25°C in 300 sec.

1µg/ml 1.0 M Hydroxyectoine 89 heat incubation Lactate Dehydrogenase T = 50°C for 30 min. 52 µg/ml 0.5 M Hydroxyectoine 90 Phosphofructokinase T = 50°C for 10 min. 75 µg/ml 1.0 M Hydroxyectoine 100 Enolase T = 50°C for 30 min. 50 µg/ml 0.1 M Hydroxyectoine 88 Carboxylesterase T = 70°C for 10 min. 200 µg/ml 2.0 M Hydroxyectoine 65 Taq Polymerase T = 95°C for 60 min. 20 IU/ml 1.0 M Hydroxyectoine 45 Monoclonal Antibody T = 60°C for 48 h 0.5 M Hydroxyectoine Active RNase A Melting (DSC) 1 mg/ml 3.0 M Hydroxyectoine Tm increase of 12 K

Lactate Dehydrogenase 52 µg/ml 1.0 M Ectoine 61 Phosphofructokinase 75 µg/ml 1.0 M Hydroxyectoine 68 enolase

freeze-drying

dT = -196°C in 30 sec. tlyophilisation = 24 h 50 µg/ml 0.4 M Hydroxyectoine 97

lactate dehydrogenase H2O2 oxidation 200 µg/ml 0.5 M Hydroxyectoine 95

Protein aggregation inhibition:

Ectoines are very effective general inhibitors of protein aggregation, as shown by their ability to inhibit the formation of amyloid fibres of Insulin and Alzheimer Beta peptide Abeta42 in vitro or the inhibition of the aggregation of LDH or some lectins.

Aggregation of LDH in Ectoine

0

0,1

0,2

0,3

0,4

0 60 120 180 240

time (min)

OD

(6

20

nm

)

Buffer + 0.5MEctoin

Buffer


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Stabilization of antibodies:

DNA Stabilization:

Protein crystallisation additive:

Stabilization of Antibodies during Freeze Thaw

Cycles

0

20

40

60

80

100

120

140

1x 2x 3x 4x

Freeze Thaw Cycles

Re

ma

inin

g A

cti

vit

y

Ki-4(scFv)-ETA in PBSKi-4(scFv)-ETA in 1 M Hydroxyectoine

Ectoines are especially suited for the protection of antibodies and antibody constructs against denaturation at high temperatures, freeze-thaw cycles and degradation.

Ectoin protects the DNA of skin cells against

damages caused by UV-irradiation

0

10

20

30

40

50

60

70

DN

A-M

igra

tio

n (

µm

)

untreated/non-irradiated

untreated/irradiated

0.014 % Ectointreated/irradiated

Ectoines can improve nucleic-acid based en-zymatic reactions (e.g. PCR). Ectoines influ-ence protein-DNA-interactions and enzymatic reactions containing DNA as a substrate, for example polymerase or hydrolase catalyzed reactions. Ectoines are also capable of protect-ing DNA from damage by chemical toxins and UV-irradiation, e.g. skin cells are protected against damages caused by UV-irradation.

Crystal Nat2 Ectoine

Beamline SLS Xo6SA-PX ESRF 1D11-1Detector MAR CD Q4R ADSC-CCDWavelength (Å) 0.980 0.934Resolution (Å) 30-2.0 (2.1-2.0) 30-1.75 (1.85-1.75)Completeness 82 (71) 96 (88)(I/σ(I)) 4.9 2.0) 11 (2.1)No. Unique observations 88947 (10357) 159267 (22268)Redundancy 3.5 (3.5) 3.6 (3.2)Rsym (%) 17 (64) 6.9 (58)

Ectoine has been used success-fully as a solvent-modifying and protein-stabilizing additive in crys-tallisation trials for X-ray structure analysis of proteins. Ectoine (100mM) improved e.g. the crystal size and quality of PAPS syn-thetase I crystals.


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Fermentation additive for the expression of functional recombinant proteins:

Many microorganisms, for example Escherichia coli, can take up Ec-toines under stress conditions from their environment to increase their stress resistance against high osmotic pressure or temperature. Extra cellular concentrations in the range between 1 – 10mM are high enough for intracellular accumulation of Ectoines under salt stress. This phenomenon can be used to increase the resistance of recombi-nant microorganisms against their highly expressed products and increase the yield of soluble protein. Inclusion body formation and

degradation during culture and down-stream processing can be reduced and recombinant pro-teins can be folded more efficiently in a native state. The result of using Ectoines as additives is an increased product yield during fermentation.

Protection of whole cells:

From the multitude of possible applications for the Ectoines some examples are listed together with the recommended Ectoine concentration.

Application Stress Recommended Concentration

Effects

Proteins

Temperature Cryostorage

Freeze-Thaw, Lyophilisation Drying

Proteolysis

0,1 - 1 M

Heat Stabilisation Freeze/Thaw Stabilisation

Maintenance of Active Conformation Chaperone Activity

Membranes Osmotic Stress Detergents 0,1% Stabilization

Protection against denaturants (SDS)

Nucleic Acids Hydrolysis Denaturation 0,1 - 100 mM Protection

Cells Dryness

Cryostorage Osmotic Stress

0,2 - 1 % Protection

1 2 3 4 51 2 3 4 51 2 3 4 5

Relative Increase of Membrane Stability of

Erythrocytes using Ectoin

0%

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

0,5% 1% 5%

Ectoin Concentration

Inc

rea

se

of

Sta

bilit

y

co

mp

are

d t

o c

on

tro

l

Cells are very sensitive to stresses like freezing or changes of the osmotic pressure. Ectoin has been shown to stabilize mammalian cell membranes, e.g. Erythrocytes. Ectoines also protect cells against UV-irradiation, chemicals (for example detergents) or dryness. Optimum conditions have to be tested out individually, but concentrations between 0.01 – 1.0 M often show best results. Ectoines do stabilize various bacteria during drying and storage. Hydroxyectoine has been suggested as a general desiccation protectant for bacterial cells


Documents

Ectoine / Hydroxyectoine - ARTIMMUN · Hydroxyectoin: C6H10N2O3, Mr 158.2, solubility in water: 4.2 M. Bulk quantities of Ectoine and Hydroxyectoine are available on request. Introduction