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Methods for crystallization of proteins, peptides and enzymes either directly from fermentation broth or during down stream processing
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Extracellular production of heme protein in fungi
Title of Technology: Methods for crystallization of proteins, peptides and enzymes either directly from fermentation broth or during down stream processing.
Abstract: This comprehensive array of crystallization technologies provides a va-riety of distinct methods for crystallizing a polypeptide or a protein, e.g. an enzyme, obtained from a protein solution, e.g. directly from a crude fermentation broth or at a later stage during down stream processing. The different methods are represented by separate patents. See below for more details.
By applying the methods of the technologies, which are simple, inexpensive and very effective, increased yields and /or purity a polypeptide or a protein, in particular an enzyme, can be obtained from even very com-plex solutions, e.g. crude fermentation broths.
Detailed Description: Enzyme crystallization using e.g. formate/acetate/nitrate (e.g. US, 5837513, US5623059, US5733764).
This technology provides a method for crystallization of enzymes, which is simple and cheap, and which is compatible to industrial requirements. It is based on an aqueous enzyme containing liquid with a relatively high enzyme purity and with a concentration of pure enzyme protein of at least 5 g/l of enzyme containing liquid as a starting material, and that a crystallization agent, which is an easily soluble salt of the non-halide type is added to the starting material to a final concentration which is smaller than the concentration needed to precipitate the enzymes in an amorphous form.
When carried out on an industrial scale, the crystals are separated in a filter, and the crystals are subsequently flushed for purification purposes. The methods based on this basic technology are simple and cheap, can be carried out with a yield of up to 95%, and that it can easily be adopted to industrial practice. The time necessary for crystallization is usually between 5 and 12 hours. By addition of crystal seeds in an amount of e.g. 1% the crystallization velocity can be accelerated. Any type of enzyme may purified, e.g. protease, lipase, amylase, cellulase, hemicellulase, pectinase, amidase or oxidase etc.
Protein crystallization using low salt and pH around pI
(e.g. US5719048)
This technlogy provide a process for the recovery of crystalline en-zymes, or potentially other proteins, which process does not require the addition of salts or organic solvents, which permits short crystalli-zation times and high yields, and which is simple and cheap, and compatible to industrial requirements. The methods, which enables the separation of an enzyme or protein from an aqueous solution comprising the enzyme in mixture with other proteins, comprises leaching out salts from the solution, adjustment of the pH of the solu-tion to a level around pI of the enzyme, and subsequent recovery of the enzyme or protein in crystalline form. Accordingly, the enzymes can be separated from even crude complex aqueous mixtures com-prising other proteins with different crystallization properties. The end result is the recovery of the desired enzyme or protein in pure, crystal-line form.
Protein crystallization using low sal t and polymer
(e.g. US5728559, US6080564) :
This technology provide a process for the recovery of crystalline pro-teins, in particular enzymes, which process does not require the addi-tion 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. It includes 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 ad-ded; and b) recovering the protein on crystalline form. If e.g. PEG is added as the water soluble polymer to the protein solution, protein with affinity to PEG will associate with the PEG molecules creating an inhomogeneous solution, which at the right PEG MW, PEG concen-tration, salt concentration and protein concentration can grow into a system consisting of a water phase with a lowered protein concentra-tion and micro droplets with a very high protein concentration. In the droplets with high protein concentration and probably with some ex-clusion of impurities, optimum conditions for crystal formation appear. The crystals then apparently take form in the droplets until they reach a size where they might burst out of the droplets generating a normal one phase aqueous system with solid crystals at the end of the crys-tallization. This proposed mechanism where the protein is crystallized in a 2-phase droplet system with the protein in a purified and concen-trated form created by the affinity between a water soluble polymer and the protein suggests that the technique can be very powerful even for low concentrated and impure protein solutions. Obviously, the technology can be applied to separation of a protein from a mix-ture of proteins, in particular to separation of an enzyme from a mix-ture of proteins, e.g. a fermentation broth.
Protein crystallization using Oxidizable salts
(e.g. US6066481) :
This technology is based on using oxidizable sulphur salts as very effective crystallization salts in low amounts on impure solutions, e.g. fermentation broths, giving short crystallization times and high yields. The technology is simple, inexpensive and environmentally friendly and provides a method for crystallization of a protein obtained from a protein solution comprising a) treatment of the protein solution with a salt containing a sulphur atom having an oxidation state less than 6, b) recovering of the protein on crystalline form. This may produce protein, e.g. enzyme, crystals from impure solutions, wherein the purity of the crystals and the yield of crystals are extraordinary good. Furthermore the morphology of the crystals may be improved compared to crystallization with other salts as the crystals are relatively large. The crystallization process according to the invention passes so quickly that normally it is not necessary to add stabilizing agents or inhibitors to the protein solution. By using the methods the harvest properties of the crystals may be improved compared with the use of other salts such as KAc; because the crystals are larger this feature makes the centrifugation and/or filtration following the crystallization much easier. If crystalline products of a very high purity are desirable, the process of the invention may be repeated, i.e. the crystalline end product of the process of the invention is redissolved and subjected to one or more additional crystallization processes.
Protein crystallization using Carbon Treatment
(e.g. US6031082) :
This technology based using a carbo n treatment prior to or especially simultaneously with a protein crystallization process increases the crys-tallization yield and crystallization purity significantly. The method of the invention can be applied directly to crude solution, e.g. a fermentation broth, or to solutions subjected to solid/liquid separatory techniques, e.g., flocculation, centrifugation, filtration, micro filtration, ultrafiltration, precipitation, evaporation, or any combination thereof. But as the method works very well on relatively impure solutions, it will normally not be necessary to purify the protein solution obtained from the fermenta-tion broth by use of chromatographic methods before the treatment with the solid adsorption material. Using this technology the crystallization yield is increased, the purity of the crystals are improved, the crystalliza-tion time for crystal formation is reduced, and the morphology of the formed crystals is changed. Thus the technology causes the polypeptide or the protein, to crystallize in a more pure form and at a higher yield. The end product may be a crystalline product or the crystals may be redissolved in order to produce e.g. a liquid polypeptide or protein prod-uct of high purity.
Patent References
| Patent Number: |
Patent: |
|
AT 96167,BE 506866,DE 69004101.2,
DK 506866,ES 50686,FR 506866,
GB 506866,IT 506866,JP 2975109
NL 506866,US 5837513,ES 631585
FR 631585,GB 631585,IT 631585
NL 631585,US 5623059,MX 191988
US 5733764
|
Enzyme cry stallization us-ing e.g. formate / acetate / nitrate |
|
AT 171189,BE 691982,CH 691982
CN 40934,DE 69413389.2,
DK 691982,ES 691982,FR 691982
GB 691982,GR 3028124,IE 691982
IT 691982,NL 691982,PT 691982
SE 691982,US 5719048
|
Protein crystallization us-ing low salt and pH around pI |
CN 55913,EP 707594A1,
US 5728559
US 6080564 |
Protein crystallization us-ing low salt and polymer
|
| EP 892810A1,US 6066481 |
Protein crystallization us-ing Oxidizable salts |
| EP 942921A1,US 6031082 |
Protein crystallization us-ing Carbon Treatment |
Title of Technology: Extracellular production of heme protein in fungi
Abstract: This technology provides a method for the extracellular production of heme proteins in filamentous fungi in yields which far exceed those obtainable for the same protein in yeast. Accordingly, the the method comprises a) transforming a suitable filamentous fungus with a recombinant DNA vector which comprises a DNA sequence encoding a heterologous heme protein, and a DNA sequence encoding a preregion permitting secretion of the expressed heme protein, and (b) culturing the transformed filamentous fungus in a suitable culture medium under conditions conducive to the production of the heme protein.
Furthermore, this technology can increase the total yield of such heme protein considerably. This is possible by adding hemin or another material containing heme groups to a fermentation medium for growing microorganisms which overproduce the apoprotein of a heme protein, the heme group is bound to the protein whereby the apoprotein is activated and acquires a conformation in which it is more stable against proteolytic degradation. In this way, endogenous heme synthesis in the host organism, which is often a bottle-neck in the expression of heme proteins, may be overcome.
Detailed Description: In this context, the term heme protein is intended to include any member of a group of proteins containing heme (e.g. protoporphyrin IX) as a prosthetic group. The term apoprotein indicates a form of the heme protein lacking the prosthetic group. The term extracellular heme protein is understood to indicate that unlike the heme proteins provided in the prior art by production in bacteria or yeast, the apoprotein for m of the heme protein is secreted from the host cell into the culture medium where it recombines (to the holoprotein) with the prosthetic heme group provided by addition of heme or heme-containing material to the medium.
The cloning and expression of varius heme proteins in bacteria has previously been described. The enzyme is expressed intracellularly as an insoluble aggregate so that it has to be purified from lysed cells. Furthermore, the enzyme is not expressed in active form and must be folded separately in the presence of heme and Ca2+ to become functional. Expression of human hemoglobins in yeast has also been described. In yeast, hemoglobin is expressed as a fully assembled, heme-containing tetramer. However, the protein is not secreted from the yeast cells, but remains in the cytoplasmic space and must be purified therefrom.
However, the technology presented here, and covered by the patents below, provides a method whereby it is possible not only to produce heme proteins but also of exporting them through the cell membrane in active form, thereby simplifying purification procedures. Further, these basic method allows for the production of heme proteins in filamentous fungi in yields which far exceed those obtainable for the same protein in yeast.
A specific process represented by one granted US patent (US5958724) consists of the following:
A process for the extracellular production of a heterologous heme pro-tein in a strain of Aspergillus sp., the process comprising:(a) transforming a suitable strain of an Aspergillus sp. with a recom-binant DNA vector which comprises a DNA sequence encoding a het-erologous heme protein, and a DNA sequence encoding a preregion permitting secretion of the expressed heme protein, and(b) culturing the tra nsformed strain of Aspergillus sp. in a suitable culture medium under conditions conducive to the production of the heme protein.
The heme protein produced by the process of the present invention could e.g. be an enzyme, e.g. an oxidoreductase such as a peroxidase, lignin peroxidase, Mn-peroxidase, or haloperoxidase.
Another important aspect of this technology is obtain significantly increased yields of protein by adding hemin or another material containing heme groups to a fermentation medium for growing microorganisms which overproduce the apoprotein of a heme protein. Hereby the heme group is bound to the protein whereby the apoprotein is activated and acquires a conformation in which it is more stable against proteolytic degradation. The total yield of heme protein is significantly increased. In this way, endogenous heme synthesis in the host organism, which is often a bottle-neck in the expression of heme proteins, may be overcome. Thus method comprises culturing a heme apoprotein producing microorganism in a fermentation medium containing heme or a heme-containing material under conditions permitting the production of active, recombined heme protein, and recovering the resulting heme protein from the medium. The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host organism in question. The heme or heme-containing material added to the medium to obtain recombination of the secreted apoprotein with the heme group may suitably be supplied by the addition of hemin or, preferably, hemoglobin or red blood cells as the heme group remains functional on heating, permitting autoclaving of media containing one of these substances.
Glossary of Terms .
| Term: |
Definition: |
| Heme protein |
heme protein is intended to include any member of a group of proteins containing heme (e.g. protoporphyrin IX) as a prosthetic group |
| Apoprotein |
apoprotein is intended to indicate a form of the heme protein lacking the prosthetic group. |
| Extracellular protein |
extracellular heme protein is understood to indicate that unlike the heme proteins provided in the prior art by production in bacteria or yeast, the apoprotein form of the heme protein is secreted from the host cell into the culture medium where it recombines (to the holoprotein) with the prosthetic heme group provided by addition of heme or heme-containing material to the medium |
| heterologous |
The term heterologous is meant to indicate proteins which are not, in nature, produced by the host organism in question. |
| filamentous fungus |
The term filamentous fungus is intended to include fungi belonging to the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes or fungi imperfecti, icluding Hyphomycetes such as the genera Aspergillus, Trichoderma, Penicillium, Fusarium or Humicola. |
&n bsp;
Patent References
Patent Number:
AT505311, BE505311, CH505311, DE505311, DK505311, ES505311, FR505311, GB505311, GR505311, IT505311, NL505311, SE505311, US5744323, US5958724,
AT180837, DE69325169.7, DK631631, ES631631, FR631631, GB631631, IT631631, NL631631, US5681725