Separation of proteins

Details Separation of proteins Separation of proteins

1995-12-01

1998-03-17

Lankford, Jr., Leon B.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

435219, 435226, 435232, 435814, C12N 900, C12N 920

Patent

active

057285594

DESCRIPTION:

BRIEF SUMMARY
TECHNICAL FIELD

The present invention relates to a method of separating a protein, in particular an enzyme, from an aqueous solution of proteins, and recovery of the desired protein on crystalline form.


BACKGROUND ART

Enzymes are usually provided as liquids or amorphous materials for industrial purposes. When not provided as liquids, they are usually provided as amorphous materials, because the known methods for crystallization of enzymes are usually regarded as too expensive to be used in an industrial scale. Due to the high purity of enzyme crystals, the provision of a cheap and simple method for crystallization of enzymes which is easily adaptable to an industrial scale is clearly a desideratum in the industry.
There is an abundance of literature concerning crystallization of enzymes. It is difficult to generalize in respect to the outcome of specific crystallization procedures. The art of enzyme crystallization is highly empirical, and a method shown to be successful for one group of enzymes does not have to be successful for other enzymes.
Characteristic features of the hitherto known protein crystallization processes are very pure and concentrated initial solutions, low yield, very long crystallization time, high consumption of chemicals including organic solvents, and poor industrial adaptability.


SUMMARY OF THE INVENTION

It is an object of the present invention to provide a process for the recovery of crystalline proteins, in particular enzymes, which process does not require the addition of large amounts of salts or organic solvents, which permits short crystallization times and high yields, and which is simple and cheap, and compatible to industrial requirements.
Accordingly, the present invention provides a method for separating a protein from an aqueous mixture of proteins comprising
(a) providing an aqueous mixture of proteins with a salt concentration at or below 1.5 Molar, to which a water soluble polymer has been added
(b) recovering the protein on crystalline form.


BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to the accompanying drawings, in which
FIG. 1 shows the peroxidase crystallization yield (%) at various PEG 4000 concentrations and various CaCl.sub.2 concentrations (.box-solid. 1% CaCl.sub.2 ; .tangle-solidup. 2% CaCl.sub.2 and .quadrature. 2.5% CaCl.sub.2).
FIG. 2 shows the peroxidase crystallization yield (%) at various pH values.
FIG. 3 shows the peroxidase crystallization yield (%) at various enzyme and PEG 4000 concentrations (.box-solid. 3% peroxidase and 1.4% other enzymes; .diamond-solid. 4.8% peroxidase and 2.3% other enzymes and .tangle-solidup. 6.7% peroxidase and 3.1% other enzymes).
FIG. 4 shows the catalase crystallization yield (%) at various CaCl.sub.2 -concentrations and at various PEG 4000 concentrations (.box-solid. 20% PEG 4000; .diamond-solid.22% PEG 4000; .tangle-solidup.24% PEG 4000; .tangle-solidup.26% PEG 4000; .diamond. 28% PEG 4000 and .increment. 30% PEG 4000).
FIG. 5 shows the catalase crystallization yield (%) at various CaCl.sub.2 -concentrations and at various PEG 4000 concentrations (.box-solid. 20% PEG 4000 and .diamond-solid. 25% PEG 4000).
FIG. 6 shows the catalase crystallization yield at various PEG 1500 concentrations and a CaCl.sub.2 -concentration of 0.2%.
FIG. 7 shows the protease crystallization yield (%) at various PEG 4000-concentrations.
FIG. 8 shows the protease crystallization yield (%) at various PEG 4000-concentrations and with a CaCl.sub.2 -concentration of 1.1%.


DETAILED DISCLOSURE OF THE INVENTION

The present invention provides a method for separating a protein or a polypeptide from an aqueous mixture comprising other proteins with different crystallization properties. It has surprisingly been found that by adding a water soluble polymer, e.g. polyethylene glycol, a protein may become separated from its mixture with other proteins and other impurities such as carbohydrate compounds, and precipitate on crystalline form.
Precipitation and crystallization of proteins with polyet

REFERENCES:
patent: 4340676 (1982-07-01), Bourque
patent: 4400471 (1983-08-01), Johal
patent: 5244800 (1993-09-01), DeLucas et al.
McPherson, A., Eur. J. Biochem., vol. 189, pp. 1-23 (1990).

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