Process for purification of mbp or mbp tagged proteins

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A process for purifying a MBP or MBP tagged protein using alginate matrix is provided herein. The process and the composition are provided for increasing the binding affinity of a target protein and recovering a higher amount of the target protein. The present invention further provides alginate matrix for purification of the MBP or MBP tagged protein. The process for purifying a MBP or MBP tagged protein using alginate matrix as disclosed in the present invention results in recovery of more than 99% of the target protein.

This invention relates generally to protein purification. In particular, the invention relates to a process for purifying a MBP or MBP tagged protein using alginate matrix and kits for use in the field of protein purification.

Production of proteins/enzymes is an important area in biotechnology. Earlier, sources for obtaining proteins were limited to animals, plants, and microorganisms. In recent decades, cloning and tissue culture techniques have been increasingly used to source proteins. Purification of proteins involves separation of the desired protein from the complex mixture of other contaminating proteins and various low molecular weight compounds present in the initial crude preparation. Purification cost can constitute sometime even upto 90% of the total production cost. Traditionally, a protein purification protocol involves a primary stage wherein unit operations like precipitation, membrane based separation separates proteins from other kinds of material, concentrates this feed from the fermenter/crude extract, and also leads to a limited purification of the desired protein. For many industrial applications, this may be an adequate level of purification. For many applications, further secondary purification stage is required. This invariably involves gel filtration, ion exchange chromatography, and affinity chromatography as the final polishing stage. For pharmaceutical proteins such as vaccines and drugs, the protein preparation of highest purity level is desirable. Over the last few decades, few clear trends have emerged in the method development area for protein purification. Reducing the number of steps (i.e. unit processes) in the purification protocol is preferred as the net protein recovery decreases as the number of steps increase. Mass transfer limitations can be avoided by carrying out the separation in the free solution rather than in the chromatographic form. This is especially a valuable strategy in the context of affinity based separations. Third trend is to develop methods which can directly deal with crude suspensions with suspended particles so that centrifugation is avoided. Aqueous two-phase system (ATPS), fluidized beds, three-phase partitioning (TPP), and macro-(affinity ligand) facilitated' three-phase partitioning (MLFTPP)are some techniques which address this issue. Magnetic separation medium are especially useful in situations wherein fermenter feed/crude homogenate has high viscosity.

Affinity based separation methods generally offer much higher selectivity as compared to separation methods based upon other principles. In any affinity based separation method, a ligand having high affinity for the target protein is linked to an insoluble (affinity chromatography) or soluble matrix (affinity precipitation, aqueous two-phase affinity extractions and MLFTPP). In fortuitous cases, a matrix is available which itself has inherent affinity for the target proteins [Gupta M.N., Guoqiang D., aul R., Mattiasson B. Purification of xylanase from Trichoderma viride by precipitation with an anionic polymer Eudragit S 100. Biotechnol. Tech. 8 . This saves the cost of the ligand and conjugation. This also leads to a safer protocol and more acceptable protocol by the regulatory agencies since the risk of ligand dissociating from the matrix during the process is eliminated. This is important if the end application of the protein is in food/pharma sectors.

Recombinant methods offer the possibility of producing the desired proteins with an 'affinity tag' (also called fusion tag) to produce the fusion protein. This enables the purification of the protein directly (skipping many steps) using an affinity based separation. The examples of popular fusion tags are polyhistidine, cellulose binding domain (CBD), Intein tag and MBP. The core of this technology is designing or selecting a suitable affinity media which would capture the fusion protein via its interaction with 'fusion tag'. MBP as a fusion tag is recommended as it is claimed that a recombinant protein produced with this tag is less likely to form "inclusion bodies". The latter are inactive solid particles often formed during overexpression of a recombinant protein in bacterial systems. This is believed to be due to aggregation of misfolded proteins. In such cases, the inclusion bodies have to be solubilized (by unfolding) and refolded to recover active soluble protein. In the case of MBP-tagged proteins, amylose , dextrin-sepharose , cationic starch , have been described as affinity media.

Using fusion tags like polyhistidine or maltose binding protein (MBP) is now a standard and widely used approach to purify recombinant proteins. The maltose-binding protein (MBP) is a 40.70 kDa, 370 amino acid, periplasmic protein of E. coli 12, involved in binding and transport of maltose and is encoded by the malE gene. The MBP can be fused at the N- or C-terminus of the protein if overexpression in bacteria is desired. For purifying MBP-tagged proteins, two affinity matrices are commercially available viz. amylose resin by New England Biolabs and Dextrin sepharose by GE Healthcare. Unlike above quoted commercially available matrices, alginate can be viewed as a smart (Ca2+ responsive), a stimuli sensitive or reversible soluble insoluble polymer. Hence a non chromatographic approach called affinity precipitation can be used for purifying proteins. Affinity precipitation is a scalable inexpensive batch process with many advantages over chromatographic methods.

US patent 5643758 describes a method for producing and/or purifying any hybrid polypeptide molecule employing recombinant DNA techniques. The method as described involves expression of hybrid polypeptide comprising a binding protein that can be purified by contacting the hybrid polypeptide with a ligand or substrate to which the binding protein has specific affinity for example affinity chromatography.

Alginate is known to bind some carbohydrate binding enzymes and can be used in affinity based separations in packed bed, fluidized bed , two phase mode and magnetic mode as well.

Thus, there is a need to produce recombinant proteins in a highly purified and well characterized form and exhibiting native conformation. In other words there is need to provide a solution for problems associated with the purification and yield of target protein. The present invention provides a solution to this problem by providing a process of purification of proteins whereby all contaminating proteins or impurities are at least partially, preferably completely, removed in a single purification step. The invention also provides for a simultaneous refolding strategy in case the starting preparation is in inactive form including inclusion bodies.