GB2473434A - Recombinant protein separation - Google Patents
Recombinant protein separation Download PDFInfo
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- GB2473434A GB2473434A GB0915735A GB0915735A GB2473434A GB 2473434 A GB2473434 A GB 2473434A GB 0915735 A GB0915735 A GB 0915735A GB 0915735 A GB0915735 A GB 0915735A GB 2473434 A GB2473434 A GB 2473434A
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- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 59
- 238000000926 separation method Methods 0.000 title claims description 15
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- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
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- 229920000057 Mannan Polymers 0.000 description 1
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- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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- General Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
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Abstract
A method for separating a recombinant protein from a cell culture comprises expressing a recombinant protein, comprising a binding domain specific to an insoluble substrate, such that the protein is released into the medium, contacting the medium with the insoluble substrate and separating or removing the complex of the substrate and recombinant protein. The recombinant protein may then be released by cleaving the binding domain from the rest of the protein. The binding domain is preferably a cellulose-binding domain. The protein may be immobilized in this way directly onto an apparatus for further analysis, such as a 96-well plate coated in cellulose. Alternatively, a porous device allowing ingress of the recombinant protein, such as a nylon bag carrying containing cellulose 1, may be inserted into the culture medium 2 to capture the recombinant protein.
Description
RECOMBINANT PROTEIN SEPARATION
This invention relates to methods and apparatus for expression and separation of recombinant proteins from cell cultures.
One of the major challenges in protein chemistry remains the design of robust steps for efficient isolation of proteins, particularly of unstable recombinant proteins, from complex culture media or fermentation broths. This downstream processing represents approximately 0-80 % of the total processing cost, and thus optimal isolation steps are a must. There is increasing demand for simple, robust and manageable technologies for isolation.
Conventional methods for cellular protein expression result in accumulation of the protein of interest in either the cytoplasm of the cell or, alternatively, in the periplasmic space.
Periplasmic expression can be controlled to give effective extracellular accumulation in the cell culture medium. Proteins expressed in this way can be recovered from the culture medium by separating cellular material from the supernatant, usually by centrifugation, and then separating the protein of interest from the culture supernatant, usually by using commercially available chromatographic or affinity purification matrices.
A recombinant protein is a protein synthesised in a recombinant cell through expression of a cloned gene. A vector, a tool for manipulating DNA, is often used to enable production of the recombinant protein from the cloned DNA sequence. Separation and purification of recombinant proteins can be a complex and time-consuming process and often a peptide or protein tag is co-expressed with the desired protein to facilitate purification. The amino acid sequence of the peptide or protein tag is often expressed at the N-terminal position of the desired protein. Additional amino acid sequences may also be co-expressed with the desired protein, such as an amino acid sequence designed to be a point of attack for a specific protease. Such a sequence could be expressed to appear between the peptide/protein tag and the desired protein such that following expression, separation and purification the tag can be easily cleaved from the desired protein, resulting in a nearly natural N-terminus sequence for the final protein. Removing the peptide/protein tag may be particularly important if the desired protein is to be used as a drug.
A peptide tag comprising an amino acid sequence of at least six histidine (His) residues is often expressed at the N-terminal region of a protein in order to isolate and purify the recombinant protein by metal chelate liquid affinity chromatography. It has been shown that an amino acid sequence consisting of 6 or more His residues will act as a metal binding site for a recombinant protein. Usually nickel ions are used as the heavy metal ion and His-tag protein is eluted from the column by histidine or imidazole.
The maltose binding protein is also often expressed at the N-terminal region of a desired protein in order to isolate and purify the protein by liquid affinity chromatography through an amylose-based column, with elution using maltose.
Though recombinant proteins are successfully separated and/or purified using centrifugation, to remove cellular material, followed by chromatography these procedures remain time consuming, and chromatography materials can be expensive. There is thus a requirement for simpler, rapid and less expensive procedures for separation and/or purification of recombinant proteins.
The present invention generally aims to provide a method for expression and separation of recombinant proteins from cell cultures, wherein the recombinant protein is expressed in a cell culture, and released into the culture medium. The recombinant protein may be released into the culture medium actively, for example it is expressed to be secreted extracellularly, or passively, for example it is released through cell death. The recombinant protein could be expressed in the periplasm of a cell such that the protein leaks into the cell medium. The applicant aims to provide recombinant proteins engineered to include a binding domain specific to an insoluble substrate, whereby the recombinant protein may be separated from the culture medium through binding to the insoluble substrate. This approach will have a number of advantages over approaches currently available to the protein chemist, and especially provides direct separation of the protein from the culture medium by simple filtration rather than by affinity chromatography.
Accordingly, in a first aspect, the present invention provides a method for expression and separation of recombinant proteins from a cell culture in a culture medium, comprising i. expressing a recombinant protein in a cell culture such that the protein is released into the culture medium, wherein the recombinant protein comprises a binding domain specific to an insoluble substrate; ii. introducing the insoluble substrate to the culture medium, thereby enabling formation of an insoluble recombinant protein-insoluble substrate complex; and iii. separating or removing the insoluble recombinant protein-insoluble substrate complex from the culture medium.
As use herein, an insoluble substrate is a substrate insoluble in aqueous solutions, such as cell culture medium. The insoluble substrate may comprise materials such as silica or agarose, or may comprise magnetic beads. The insoluble substrate may comprise an element of a diagnostic apparatus, such as the gold surface of a surface plasmon resonance sensor or a plastic 96-well plate. Such materials may be coated with a ligand specific to the binding domain. Such ligands may include proteins, such as antibodies or lectins, or carbohydrates.
The insoluble substrate may be of biological origin, such as an insoluble protein or carbohydrate. The insoluble substrate is preferably inert.
In a preferred embodiment the insoluble substrate is an insoluble carbohydrate, such as insoluble starches, mannan, chitin or xylan. In a more preferred embodiment the insoluble substrate is the carbohydrate cellulose. Cellulose is a polysaccharide comprising glucose.
The glucose units are linked via 13-glycosidic linkages from the anomeric position (C-i) of one glucose monosaccharide unit to the 4-position (C-4) of the next glucose monosaccharide unit. The configuration of the anomeric carbon (i.e. 13) results in cellulose chains being essentially linear. The position of the hydroxyl groups in a cellulose chain enables multiple cellulose chains to bind to each other via hydrogen bonding chains resulting in a highly insoluble and rigid polymer. Cellulose is inexpensive, inert, non toxic and non metabolisable.
The binding domain specific to the insoluble substrate is a peptide or protein capable of specifically binding to the insoluble substrate, for example the binding domain may be an antibody or fragment thereof specific to the insoluble substrate. In the embodiment wherein the insoluble substrate is a carbohydrate the binding domain is a carbohydrate binding domain, such as a lectin or fragment thereof that specifically binds the carbohydrate.
Wherein the insoluble substrate is cellulose the binding domain is a synthetic or natural cellulose binding domain (often called a cellulose binding module). Cellulose binding domains found in nature, especially fungi and bacteria, are discrete domains from proteins such as cellulases. Over 120 cellulose binding domain amino acid sequences have been identified, and these have been classified into eleven families. Cellulose binding domains are well known in the art.
In a preferred embodiment of the first aspect the step of introducing the insoluble substrate to the culture medium is performed prior to or simultaneously to the step of expressing the recombinant protein in the cell culture. The presence of the insoluble substrate in the cell culture medium from the beginning of the expression enables continuous production of the insoluble recombinant protein-insoluble substrate complex throughout the period of expression. The presence of insoluble substrate in the medium throughout expression is believed to positively affect the equilibrium of extracellular production such that the yield of recombinant protein is higher than that in the absence of insoluble substrate. The insoluble substrate may however be introduced to the cell culture at any point during or after expression.
In a preferred embodiment the method further comprises isolating the recombinant protein from the insoluble recombinant protein-insoluble substrate complex. This may be achieved by, for example, treatment of the complex with an acidic or basic solution, a high molarity salt solution, or a more specific inhibitor of the interaction, such as a methyl glycoside for the interaction of a carbohydrate to a carbohydrate binding domain.
In a more preferred embodiment the recombinant protein further comprises an amino acid sequence capable of cleavage by a protease. In this embodiment, the method further comprises cleaving/removing the binding domain from the recombinant protein through use of the protease. Cleaving of the binding domain from the recombinant protein is particularly important when the desired protein is required in a natural or nearly natural form, such as may be required if the protein is to be used as a drug.
The cell culture is preferably a bacterial cell culture, for example the recombinant protein could be expressed in Escherichia coli. The culture medium comprises reagents suitable for growth of the cells and expression of the recombinant protein which are known and understood by the person skilled in the art.
The step of separating or removing the insoluble recombinant protein-insoluble substrate complex may be achieved by filtration or decantation from the culture medium, for example the insoluble complex may be separated from the culture medium, including the cells, by simple filtration through a course sieve. The insoluble substrate may then be washed, before the protein is eluted from the insoluble substrate.
In a preferred embodiment, the method comprises introducing the insoluble substrate to the culture medium in a porous device. The protein may then be removed from the culture medium by removing the porous device from the culture medium. The porous device allows ingress of the recombinant protein from the culture medium, but preferably not the cellular material. The porous device is preferably provided with means for introducing and removing the device from the cell culture. Suitable porous devices include nylon mesh bags, such as those used as tea bags. The use of a porous device enables easy removal of the insoluble substrate from the culture medium without the need for filtration or decanting.
Embodiments of the method wherein the insoluble substrate comprises an element of a diagnostic or detection apparatus, such as a 96-well plate, are particularly advantageous since the recombinant protein is rapidly made available for utilisation in a diagnostic assay or in detection of a target molecular species without any ifirther preparation or modification of the element of the apparatus. Many diagnostic and detection apparatus require proteins to be bound to the surface of an element of an apparatus. This method enables the protein to be bound to a surface of an element of an apparatus directly from the cell culture, without the need for any further processing steps.
The method of the first aspect is more efficient, faster, cheaper and potentially safer to the individual and the environment (for example, cellulose is non-toxic), than currently used methodologies.
In a second aspect, the present invention provides a kit for expression and separation of recombinant proteins from a cell culture in a culture medium, comprising reagents for expressing a recombinant protein comprising a binding domain specific for an insoluble substrate, and the insoluble substrate. The reagents preferably include a vector for expressing the recombinant protein comprising the binding domain.
In a preferred embodiment the kit further comprises a porous device capable of allowing ingress of the recombinant protein, but preferably not the cellular material. The porous device is preferably a nylon mesh bag.
In an alternative preferred embodiment the insoluble substrate comprises an element of a diagnostic or detection apparatus. The element may for example be a 96-well plate.
Alternatively, the kit for expression and separation of recombinant proteins from a cell culture in a culture medium may comprise the porous device and the insoluble substrate, and optionally reagents for expressing a recombinant protein comprising a binding domain specific for an insoluble substrate.
The insoluble substrate is preferably a carbohydrate, and most preferably cellulose. In the embodiment wherein the insoluble substrate is a carbohydrate the binding domain is a carbohydrate binding domain, and wherein the insoluble substrate is cellulose the binding domain is a cellulose binding domain.
The present invention will now be described with reference to the following non-limiting examples and drawings in which Figure 1 is a schematic of one embodiment of the invention representing a culture flask, a culture medium and a nylon mesh bag, comprising insoluble substrate, suspended in the culture medium; Figure 2 is a photograph of an SDS PAGE gel comparing culture supematant (lane A), eluant from insoluble cellulose in a 0.5 micron mesh nylon bag (Lanes B and C), eluant from insoluble cellulose in a 1.0 micron mesh nylon bag (Lanes D and E), purified ovalbumen-kappa recombinant protein (0.5 tg and 1.0 tg; Lanes F and G), and molecular weight standards (Lane H). The major protein present in lanes B-E (circled) is the ovalbumen-cellulose binding domain fusion antibody with a predicted molecular weight of 43.6 KDa.
The predicted size of the ovalbumen-kappa fusion is 38 KDa; and Figure 3 is a graph representing data from an ELTSA of purified ovalbumen-cellulose binding domain fusion antibody eluted from cellulose. Purified protein (2ug, lug and O.5ug) bound specifically to ELISA plate wells coated with hen egg ovalbumen.
Examples
Example 1
Recombinant protein tagged with a cellulose binding domain may be extracellularly expressed from a recombinant bacterial species following insertion of the relevant cloned DNA sequence(s). Cellulose, a biological polymer insoluble in aqueous solvents, could then be added directly to the cell culture medium whereby the expressed protein would bind to and accumulate on the surface of the cellulose. One result of binding the expressed protein to the cellulose is that the concentration of the protein in the culture medium will decrease, thereby affecting the equilibrium of the system, and potentially resulting in an increase in is protein yield as compared to expression in the absence of cellulose from the culture medium.
Once the expression has been completed the cellulose can be separated from the culture medium and cells by simple filtration through a course sieve. The cellulose may then be washed to remove any non-specifically bound proteins, and finally the recombinant protein eluted from the cellulose through use of a methyl-glycoside or free sugar.
Example 2
A recombinant antibody specific to ovalbumen was expressed to contain a cellulose binding domain covalently attached to the N-terminus of the antibody. Expression was in Escherichia coli in liquid broth. Having regard to Figure 1, the experimental setup included immersing a 0.5 micron or 1.0 micron nylon bag 1 comprising insoluble cellulose (acid washed cellulose) into the culture medium 2 prior to expression. Having regard to Figure 2, and lanes B to E, the recombinant fusion antibody was secreted into the culture medium during the expression, and migrated through the pores of both the 0.5 and 1.0 micron nylon bag, and bound to the cellulose. The major protein eluted from the cellulose had the expected molecular weight for the antibody-cellulose binding domain product (43.6 kDa) and having regard to Figure 3 was shown to bind hen egg ovalbumen. The yield of protein isolated was at least equal to that produced by known isolation methods.
In principal any binding substrate larger than the pore size of the bag could be used to facilitate isolation and purification of extracellularly expressed recombinant proteins. The binding substrate could for example include ligands attached to agarose or magnetic beads.
The binding occurs in the culture medium and in this example isolation is achieved successfully from a complex culture media, i.e. liquid broth.
Claims (20)
- CLAIMS1. A method for expression and separation of recombinant proteins from a cell culture in a culture medium, comprising i. expressing a recombinant protein in a cell culture such that the protein is released into the culture medium, wherein the recombinant protein comprises a binding domain specific to an insoluble substrate; ii. introducing the insoluble substrate to the culture medium, thereby enabling formation of an insoluble recombinant protein-insoluble substrate complex; and iii. separating or removing the insoluble recombinant protein-insoluble substrate complex from the culture medium.
- 2. A method according to Claim 1, in which step of introducing the insoluble substrate to the culture medium is performed prior to or simultaneously to expressing the recombinant protein in the cell culture.
- 3. A method according to Claim 1 or Claim 2, further comprising isolating the recombinant protein from the insoluble recombinant protein-insoluble substrate complex.
- 4. A method according to Claim 3, ftirther comprising cleaving the binding domain from the recombinant protein,
- 5. A method according to Claims ito 4, in which the cell culture is a bacterial cell culture.
- 6. A method according to Claims 1 to 5, wherein the insoluble substrate is introduced to the culture medium in a porous device and the insoluble recombinant protein-insoluble substrate complex is separated or removed from the culture medium by removing the porous device from the culture medium, wherein the porous device allows ingress of the recombinant protein.
- 7. A method according to Claims 1 to 5, wherein the insoluble substrate comprises an element of diagnostic or detection apparatus.
- 8. A method according to Claim 7, in which the element is a 96-well plate.
- 9. A method according to Claims 1 to 8, in which the insoluble substrate is an insoluble carbohydrate.
- 10. A method according to Claim 9, in which the insoluble carbohydrate is cellulose.
- 11. A method according to Claim 10, in which the binding domain is a cellulose binding domain.
- 12. A kit for expression and separation of recombinant proteins from a cell culture in a culture medium, comprising reagents for expressing a recombinant protein comprising a binding domain specific for an insoluble substrate, and the insoluble substrate.
- 13. A kit according to Claim 12, in which the reagents comprise a vector for expressing the recombinant protein comprising the binding domain.
- 14. A kit according to Claim 12 or Claim 13, further comprising a porous device, wherein the porous device allows ingress of the recombinant protein.
- 15. An kit according to Claim 12 or Claim 13, in which the insoluble substrate comprises an element of a diagnostic or detection apparatus.
- 16. A kit according to Claim 15, in which the element is a 96-well plate.
- 17. A kit according to Claims 12 to 16, in which the insoluble substrate is cellulose.
- 18. A kit according to Claim 17, in which the binding domain is a cellulose binding domain.
- 19. A kit for expression and separation of recombinant proteins from a cell culture in a culture medium substantially as hereinbefore described with reference to the examples and is figure 1.
- 20. A method for expression and separation of recombinant proteins from a cell culture in a culture medium substantially as hereinbefore described.
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GB0915735A GB2473434A (en) | 2009-09-09 | 2009-09-09 | Recombinant protein separation |
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GB0915735A GB2473434A (en) | 2009-09-09 | 2009-09-09 | Recombinant protein separation |
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GB2473434A true GB2473434A (en) | 2011-03-16 |
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5738984A (en) * | 1993-04-14 | 1998-04-14 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Kits and methods of detection using cellulose binding domain fusion proteins |
US6048715A (en) * | 1988-07-08 | 2000-04-11 | Univ. Of British Columbia | Two-phase partition affinity separation system and affinity separated cell-containing composition |
WO2003018797A2 (en) * | 2001-08-22 | 2003-03-06 | Helix Biopharma Corporation | Method and device for integrated protein expression, purification and detection |
WO2005023981A2 (en) * | 2003-09-09 | 2005-03-17 | Zephyr Proteomix Ltd. | Libraries of chimeric cellulose binding proteins and methods of use thereof |
US20070065880A1 (en) * | 2005-09-20 | 2007-03-22 | New England Biolabs, Inc. | Compositions and methods relating to elutable carbohydrate-binding proteins |
-
2009
- 2009-09-09 GB GB0915735A patent/GB2473434A/en not_active Withdrawn
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6048715A (en) * | 1988-07-08 | 2000-04-11 | Univ. Of British Columbia | Two-phase partition affinity separation system and affinity separated cell-containing composition |
US5738984A (en) * | 1993-04-14 | 1998-04-14 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Kits and methods of detection using cellulose binding domain fusion proteins |
WO2003018797A2 (en) * | 2001-08-22 | 2003-03-06 | Helix Biopharma Corporation | Method and device for integrated protein expression, purification and detection |
WO2005023981A2 (en) * | 2003-09-09 | 2005-03-17 | Zephyr Proteomix Ltd. | Libraries of chimeric cellulose binding proteins and methods of use thereof |
US20070065880A1 (en) * | 2005-09-20 | 2007-03-22 | New England Biolabs, Inc. | Compositions and methods relating to elutable carbohydrate-binding proteins |
Non-Patent Citations (2)
Title |
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Biootechniques (1006); Vol 20, pp 616-620, "Use of a 96-well format...", Felleisen et al * |
Bioseparation (1995); Vol 5, pp 95-104, "Purification of human interleukin-2...", Ong et al * |
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