Within the general field of biotechnology, the ability to effectively separate and purify proteins of commercial value from complex sources such as living cells, blood serum, or fermentation broth is of critical importance. Applications in industry and research laboratories where purified or partly purified proteins are used are numerous and well documented in prior literature. See, for example, D. R. Meadon and G. Walsh in Biotechnological Advances 1994, 12: pp. 635-646.
The majority of currently employed protein purification techniques exploit the innate physical and chemical properties between proteins. One method of purifying proteins are the affinity-based purification techniques, which are unique in that, rather than relying on relatively nonspecific differences in physicochemical properties of proteins, they capitalize on the highly specific biological recognition between two molecular species which form an affinity pair. Binding of the two entities of the affinity pair occurs in almost all instances as a result of relatively weak chemical interactions, known as non-covalent bonds. Some recognized and commonly used affinity pairs include antibodies and their binding antigenic substances, nucleic acid binding proteins and nucleic acids, lipid binding proteins and lipids, lectins and carbohydrates, streptavidin/biotin complexes, protein A/immunoglobulin G complexes, and receptors and their binding substances.
In general, affinity based purification processes require that one member of the affinity pair is immobilized on a solid substrate or matrix that is insoluble in the fluid in which the other member of the pair resides. It is a further requirement that immobilization is accomplished in such a fashion that the capacity of the members of the affinity pair to recognize each other is not adversely affected by the immobilization procedure. The molecular species of the affinity pair bound to the matrix is generally referred to as the ligand, while the liquid soluble member is generally referred to as the target member. It is important to note that these definitions do not impose any restrictions in a strict chemical sense.
The vast majority of current immobilization techniques rely on physical or chemical approaches. Physical ligand immobilization involves adsorption or entrapment of the ligand to a suitable support, while the chemical mode of immobilization is characterized by the formation of strong crosslinks or covalent attachments between the ligand and the matrix.
It is a disadvantage of the currently available physical and chemical techniques for immobilizing ligands that production processes are frequently time consuming and expensive. This is mainly due to the fact that immobilization techniques require the separate production of matrix material and ligands, which in a subsequent step must be coupled. An alternative mode of immobilizing proteins is described in U.S. Pat. No. 5,474,925 which documents a biological production system for the immobilization of enzymes in the fibre of cotton plants. This patent discloses what is believed to be the first biologically produced enzyme immobilization system and allows a one step production of matrix and ligand.
Subsequent to immobilization of the ligand on the matrix, a variety of affinity based purification techniques may be employed to accomplish selective binding between the ligand and the target member. In the most widely used affinity based purification technique, affinity chromatography, a matrix containing a ligand is coated to, or packed on, the inside of a chromatographic column. A complex mixture containing the target member is then applied to the chromatographic column. Ideally, only the target molecules that specifically recognize the ligand bind in a non-covalent fashion to the chromatographic column, while all other molecular species present in the sample pass through the column. Other affinity-based purification technologies known to the prior art include, perfusion affinity chromatography, affinity repulsion chromatography, hyperdiffusion affinity chromatography, affinity precipitation, membrane affinity partitioning, affinity cross-flow ultrafiltration and affinity precipitation.
In affinity partitioning two solutions of substantially different densities are employed. The complex solution containing the target member is mixed with a solution of a different density containing the affinity ligand. Subsequent to mixing, the solutions are left to settle in order to permit the formation of two separate phases. Molecules tend to partition differentially between phases depending on their size, charge and specific interactions with the phase-forming solutions. Ligand-bound target protein selectively partitions to the phase containing the affinity ligand. For example, Coughlin and Baclaski in Biotechnology Progress, 1990 6: 307-309 reported the use of the biotin containing organic solution isooctane to transfer avidin from an aqueous solution to the isooctane solution. However, so far applications of affinity partitioning have been limited mainly due to the current lack of availability of suitable affinity matrix substances which can be employed in specific partitioning in two phase systems.
An important factor for the commercial development of biotechnology is the purification of bioproducts, which typically accounts for 50% or more of the total costs. Many purification steps rely on column or batch type separation procedures. In particular large scale high-separation techniques such as column chromatography affinity based purification techniques are costly. In addition, crude material is less suitable for either batch purification or column chromatography, as contaminants may foul up sedimented resins and plug columns. Thus, affinity matrices are often only employed in a later stage of purification processes where substantial purity is critical, where the proteins are present in extremely dilute concentrations, and/or where high value proteins are required, for example in diagnostic and therapeutic proteins. These and other topics related to the use of affinity technology in biotechnological processes have been reviewed by Labrou, N. and Clonis, Y. D. in the Journal of Biotechnology 36: 95-119 (1994).
There is a need in the art to develop a method for purifying biological products from complex mixtures. The present inventors have found that oil bodies and their associated proteins are useful in this regard.
Oil bodies are subcellular oil storage structures. Typically oil bodies accumulate in the seeds of higher plants and are enveloped by a phospholipid monolayer into which are embedded a limited amount of proteins referred to as oil body proteins. Oleosins are the predominant species of plant oil body protein present in the oil body phospholipid monolayer. Structurally oleosins are characterized in that they contain a highly lipophilic central portion which serves to anchor the protein in the phospholipid monolayer and more hydrophillic N- and C- termini which reside in the cytoplasm.
PCT application Publication No. WO 96/21029 describes the use of oil bodies for the preparation of recombinant proteins in plants. In this technology, the gene encoding the foreign protein is genetically fused to a plant oleosin gene, resulting in a fused protein product. Following biosynthesis the fusion protein is targeted to the oil body fraction of plant seeds. Floatation centrifugation is used to separate oil bodies from the aqueous portion of the seeds.
The present inventors have found that both oleosins fused to recombinant proteins as well as native oleosins are useful as affinity matrices.