Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Reexamination Certificate
1995-07-24
2001-01-16
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
C435S069100, C435S069700, C435S070100, C435S071100, C435S071200, C435S173300, C435S178000, C435S179000, C435S320100, C435S803000, C435S815000, C436S529000, C436S530000, C530S413000, C530S813000, C530S814000
Reexamination Certificate
active
06174700
ABSTRACT:
INTRODUCTION
1. Technical Field
This invention relates to methods for separating and/or concentrating polypeptides and other compounds by affinity phase separation using a polymer-ligand pair in which the ligand binds to soluble phase-forming oligosaccharides. The invention is exemplified by the use of a phase separation system comprising a soluble oligosaccharide with affinity for a compound comprising as the affinity ligand a cellulose-binding domain from a
Cellulomonas fimi
cellulase.
2. Background
Production of proteins by expression in microbial systems has become a significant source of high value, medically important proteins. Purification and recovery of recombinant proteins are major considerations in the design of a fermentation process. While traditional means of protein purification can be used to isolate a product, more recently, aqueous two-phase extraction systems have received considerable industrial interest as a means to simplify large-scale purification of protein products, including high-dose therapeutics such as insulin, and industrial proteins such as 3-oxosteroid isomerase, alcohol dehydrogenase, and phosphofructokinase. As a result, a wide variety of two-phase systems are now available for both protein-purification and cell-separation applications. Extraction in aqueous two-phase systems offers unique advantages for large-scale processing of recombinant proteins and peptides, including high activity yields (i.e., the largely aqueous environment minimizes protein inactivation during purification), fast approach to equilibrium, easy scale-up and, most importantly, continuous processing. Technical feasibility of aqueous two-phase partition systems has been demonstrated in several systems up to the 100,000 L scale. They are formed by adding to water either two water-soluble but incompatible polymers or a water-soluble polymer and a strong electrolyte. Polyethylene glycol (PEG) serves as one of the polymer components in many industrial two-phase systems due to its low price and availability in a wide range of molecular-weight fractions. Fractionated dextran, an &agr;-1,6 glucosaccharide with &agr;-1,3 branching, often serves as the second polymer. However, many other water-soluble polymers are also in use, including a number of other carbohydrates. All aqueous two-phase systems contain mainly water, with each phase enriched in one of the separation-inducing components and nearly devoid of the other. When a mixture of proteins and other biomacromolecules from a fermentation broth is added to an aqueous two-phase system, each type of protein partitions uniquely based on its relative affinity for the two phase-forming components, as well as on its size, surface chemistry, and net charge.
Relatively low partition coefficients and lack of selectivity in conventional aqueous two-phase systems have motivated the development of affinity partition systems which combine the versatility of conventional partition systems with the unique binding selectivities of affinity ligands. In most cases, the biospecific ligand is covalently linked to one or both ends of a phase-forming polymer, usually PEG. The strong partitioning of the polymer during phase formation then causes the accumulation of ligand into one of the equilibrium phases. This highly asymmetric partitioning, combined with the strong affinity of the target protein for the ligand, is the basis behind the affinity separation and concentration.
However, although they are finding some industrial use, current affinity partition systems are limited in their capacity and resolving power by low ligand densities which result from the presence of only one or two ligands per polymer chain. Since polymer concentrations are usually less than 15 wt %, affinity partition systems with a 1:1 or 2:1 ligand to polymer stoichiometry usually yield target protein separation factors (relative to those of the contaminants) between 5 and 50. While these separation factors are more than sufficient for product concentration, they do not generally provide a desired product purity in a cost-effective, one or two-stage extraction process. Classic affinity partition systems are also limited by the expense of the chemistry needed to produce the polymer-ligand conjugates. For instance, conjugation of a ligand to PEG first requires substitution of the terminal hydroxyl groups with more reactive electrophiles, such as bromides, chlorides, or epoxides. A second nucleophilic-attack reaction is then required to covalently bind the polymer and ligand. The ligand polymer conjugates also must be designed specifically for each protein or class of proteins to be purified. It therefore is of interest to develop rapid, inexpensive, high capacity methods for purification of a desired protein, particularly to develop methods which can use generic polymer-ligand conjugates.
Relevant Literature
References relating to endoglucanase C include the following. Moser et al.,
Applied and Environmental Microbiology
(1989) 55:2480-2487
; Molecular Microbiology
(1991) 5:1221-1233; Coutinho, et al.,
Molecular Microbiology
(1992) 6:1243-1252; and Coutinho, et al.
FEMS Microbiology Letters
(1993) 113:211-218. For a review of &bgr;-1, 4-glycanases, see Gilkes, et al. (1991)
Microbial Reviews
55:303-315. Also, see Miller, Jr., et al. (1995)
Proc
. 6
th Int. Conf. on Biotechnology in the Pulp and Paper Industry
, Vienna, Austria.
SUMMARY OF THE INVENTION
Aqueous phase separation and/or purification systems, together with methods for their preparation and use, are provided which are based on polymer-ligand conjugates wherein the polymer is an oligosaccharide polymer and the composition to be separated and/or purified comprises a ligand which binds to the oligosaccharide polymer. The ligand is a polysaccharide binding peptide (PBP) which is an amino acid sequence characterized as capable of binding to a phase-forming oligosaccharide polymer. The composition generally is a fermentation broth, a biological fluid, or other fluid containing a compound comprising a macromolecule or chemical moiety of interest fused to a PBP. The phase separation system includes the oligosaccharide polymer and a phase-inducing polymer or other phase-inducing agent. The method involves contacting the phase separation system with the composition, which partitions into the oligosaccharide polymer phase, and isolating the composition. The composition may be removed from the oligosaccharide polymer with a removal solution having low ionic strength, high pH or containing a chaotropic agent. Alternatively, a specific or non-specific protease can be used for enzymatic removal of the compound from the polysaccharide binding moiety which remains bound to the oligosaccharide polymer by incorporating a protease recognition sequence between the compound and the polysaccharide binding moiety. Where a protease is used, it can be provided bound to a second polysaccharide binding moiety having affinity for a crystalline polysaccharide to which the first polysaccharide binding moiety has no affinity. Optionally, the protease can be recycled by subsequent elution from the solid polysaccharide. Alternatively, the protease bound to the second polysaccharide binding moiety can be provided bound to a solid polysaccharide support to which the polysaccharide binding peptide does not bind. The invention finds use for separation and/or purification of proteins and other compounds.
REFERENCES:
patent: 5202247 (1993-04-01), Kilburn et al.
patent: 5340731 (1994-08-01), Kilburn et al.
Coutinho et alMolecular Microbiology(1992) 6(9):1243-1252.
Greenwood et alBiotechnol. and Bioeng.(1994) 44:1295-1305.
Johansson et alCA:—98193917.
Ong et alCA:122:312495.
Skuse et alEnzyme Microb. Technol.(1992) 14(10):785-790.
Wierzba et alBiotechnol. And Bioeng.(1995) 47:147-154.
Coutinho et al.,FEMS Microbiol. Lett.(1993) 113:211-218.
Coutinho et al.,Mol. Micobiol.(1991) 5:1221-1233.
Gilkes et al.,Microbiol. Rev.(1991) 55:303-315.
Graham et al.,Gene(1995) 158:51-54.
Graham et al.,Nucleic Acids Res.(1993) 21:4923-4928.
Miller et al.,
Haynes Charles A.
Kilburn Douglas G.
Tomme Peter
Naff David M.
Rae-Venter Barbara
Rae-Venter Law Group P.C.
University of British Columbia
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