Zbasic—A novel purification tag for efficient protein recovery

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Abstract

A positively charged protein domain, Zbasic, can be used as a general purification tag to achieve efficient recovery of recombinantly produced target proteins using cation-exchange chromatography. To construct a protein domain usable for ion-exchange chromatography, the surface of protein Z was engineered to be highly charged, which allowed for selective capture of target proteins on a cation-exchanger at physiological pH values. Interestingly, the novel domain, denoted Zbasic, was shown to be selective also under denaturing conditions and could preferably be used for purification of proteins solubilised from inclusion bodies. Moreover, a flexible process for solid-phase refolding was developed, using Zbasic as a reversible linker to the cation-exchanger resin. This procedure has the inherited advantage of combining purification and refolding into a single step and still enabling elution of a concentrated product in a suitable buffer. This article summarizes development and use of the Zbasic tag in small and pilot-plant-scale downstream processing.

Introduction

High throughput recovery of recombinant proteins desires a general approach that uses a minimum of unit operations during the procedure but still yields a predictable result. The use of purification tags has become a major breakthrough since it contributes with a selective property to each target protein, which generalises and facilitates the recovery process. A large repertoire of purification tags is now available [1] and each tag has its own characteristics; advantages and disadvantages. However, most often, a desired high selectivity obligates harsh conditions to release the protein from the affinity matrix, which can affect the protein of interest. For large-scale processes it is also of great importance that the resin used is stable for all conditions applied during the process, including the cleaning steps. Most affinity chromatography resins are based on protein ligands and are thus quite fragile. An ideal purification system would combine high selectivity, a robust and reusable matrix and retained possibility of mild and efficient elution.

For purification of native proteins, one of the most widely used techniques is ion-exchange chromatography (IEX). This is mainly due to the advantageous properties of the IEX resin, which provides high capacity, good resolution and a robustness that enables reusability. Moreover, due to high production quantities and the simplicity of the ligand, these resins are relatively cheap [2]. The main drawback of IEX is that each time a new protein is to be purified the method has to be optimized according to the individual charge pattern of that specific target protein. An attractive strategy is to fuse a charged moiety to the recombinant protein, thus allowing advantages from affinity systems to be combined with the usage of the favourable IEX resin. Exploiting this principle, peptides containing highly charged amino acids have been used as purification tags in order to recover proteins selectively by IEX [3], [4], [5], [6], [7]. Unfortunately, most systems based on charged peptides suffer from drawbacks such as loss of expression and proteolytic instability.

Apart from increasing the selectivity of a protein by the incorporation of a charged peptide, it is also possible to engineer the target protein to include a larger number of charged amino acids [8]. However, this strategy is laborious, since it requires engineering of each individual target protein and also analysis of the resulting mutant to ascertain that its function has not been changed. However, it is also possible to endow proteins tags with desired properties by protein engineering methods. This allows for tailor making of tags to suit a particular production and purification scheme.

Section snippets

Molecular engineering of a charged purification tag

In order to create a universal purification handle for ion-exchange chromatography, a domain with highly charged surface was developed by protein design (Fig. 1) [9]. Since most proteins in Escherichia coli (E. coli), one of the most frequently used host organism for recombinant protein production, have a neutral or low isoelectric point [10], the tag was engineered to contain multiple positive charges to be able to interact strongly with a cation-exchanger. By taking advantage of a stable

Analysis of Z variants

In order to characterize the secondary structure of the different Z variants, their absorption of far-ultraviolet circular polarized light was measured. Circular dichroism (CD) spectra collected for the starting scaffold reveal a molecule with an alpha-helical structure, which also is in accordance with the determined NMR-structure of the Z domain [23]. Despite the number of positively charge residues introduced, spectra for the basic Z variants show a similarity that implies that they have a

Zbasic as a general purification tag

To evaluate the usefulness of Zbasic as a purification tag, it was investigated if its chromatographic properties were preserved also after fusion to a target protein [24]. As a model target protein, the Klenow fragment, an exonuclease deficient variant of E. coli DNA-polymerase I, was chosen. A purification process is preferably performed at pH 7.5, since most proteins, including Klenow, has maximally retained bioactivity at physiological pH. However, fusion with the rather large (86 kDa) and

Integrated process for selective protein recovery

The promising results achieved with Zbasic encouraged the development of a pilot-plant-scale purification strategy. An optimal process operates under physiological conditions and provides high amounts of a pure and native product by the use of as few steps as possible. The development of an integrated strategy for production, purification, efficient cleavage and subsequent removal of the purification tag was considerable helped by the positively charged tag.

In order to obtain a high production

Immobilised protease for cleavage of fusion proteins

The Zbasic tag facilitated manufacturing of the protease and also its removal after completed cleavage (Fig. 3A). However, a new batch of protease has to be produced each time a substrate is to be cleaved. To further improve the process and to enable reusage of the protease, a set-up with directed immobilisation of the protease on to a solid support in a column was developed, as illustrated in Fig. 3B. Having the protease fused to a tag can also be advantageous for subsequent immobilisation of

The impact of Zbasic on protein solubility

One commonly used strategy to increase solubility is to express the target protein with a protein that has high solubility, e.g. thioredoxin and MBP [30]. Z, the parental domain to Zbasic, is highly soluble and has also been shown to increase the solubility of fusion proteins that otherwise are insoluble [15]. In order to evaluate the effect that Zbasic has on the expression pattern, a novel flow cytometry-based screening method was used [31]. A set of 20 different protein fragments were chosen

Purification under denaturing conditions

In cases when the target protein is produced as inclusion bodies, the property used for separation must be selective under denaturing conditions. To explore the suitability of Zbasic as a tag for purification of inclusion body proteins, the capability of Zbasic to bind the cation-exchange matrix under denaturing conditions was investigated [26]. In 8 M of urea, the Zbasic moiety could still be completely adsorbed to a cation-exchanger at pH 7.5. However, the interaction was less strong, as Zbasic

Solid-phase refolding of Zbasic tagged fusion proteins

For most purposes, the native structure of a protein is an obligate. Accordingly, purification of inclusion body proteins has to be followed by in vitro refolding. Proteins tagged with poly-cationic peptides have previously been shown to be successfully refolded using solid-phase strategies [33], [34]. The fact that Zbasic could be captured on a cation-exchanger under denaturing conditions and also regain the identical adsorption characteristics as the native protein upon complete removal of

Concluding remarks

Here, we have summarized the results and strategies for the development of a novel purification tag, Zbasic. This protein domain was constructed to have a highly charged surface and was shown to be suitable for general use as fusion partner to different target proteins. The strategy combined the selectivity of affinity purification with the convenience and cost-effectiveness of ion-exchange chromatography. The Zbasic tag have been shown to be highly selective under native conditions, still the

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