Mutation of surface-exposed histidine residues of recombinant human granulocyte-colony stimulating factor (Cys17Ser) impacts on interaction with chelated metal ions and refolding in aqueous two-phase systems

Site directed mutagenesis of Cys17-->Ser17 form of recombinant human granulocyte colony stimulating factor (rhG-CSF C17S) for sequential replacing of surface His(43) and His(52) with alanine was used to identify residues critical for the protein interaction with metal ions, in particular Ni(2+) chelated by dye Light Resistant Yellow 2 KT (LR Yellow 2KT)-polyethyleneglycol (PEG), and refolding after partitioning of inclusion bodies in aqueous two-phase systems. Strong binding of rhG-CSF (C17S) to PEG-LR Yellow 2KT-Cu(II) complex allowed for the adoption of affinity chromatography on Sepharose-LR Yellow 2KT-Cu(II) that appeared to be essential for the rapid isolation of mutated forms of rhG-CSF. Efficiency of that purification stage is exemplified by isolation of rhG-CSF (C17S, H43A) and rhG-CSF (C17S, H43A, H52A) mutants in correctly folded and highly purified state. Affinity partitioning of rhG-CSF histidine mutants was studied in aqueous two-phase systems containing Cu(II), Ni(II) and Hg(II) chelated by LR Yellow 2KT-PEG at pH 7.0 and Cu(II)-at pH 5.0. It was determined, that affinity of rhG-CSF mutants for metal ions decreased in the order of C17S>C17S, H43A>C17S, H43A, H52A for Cu(II), and C17S=C17S, H43A>C17S, H43A, H52A for Ni(II) ions, while affinity of all rhG-CSF mutants for Hg(II) ions was of the same order of magnitude. Influence of His(43) and His(52) mutation on protein refolding was studied by partitioning of the respective inclusion body extract in aqueous two-phase systems containing Ni(II) and Hg(II) ions. Data on rhG-CSF histidine mutant partitioning and refolding indicated, that His(52) mutation is crucial for the strength of protein interaction with chelated Ni(II) ions and refolding efficiency.     

Aqueous two-phase metal affinity extraction of heme proteins

An iminodiacetic acid derivative of poly(ethylene glycol) (PEG-IDA) that chelates metal cations has been synthesized and used to extract proteins in metal affinity aqueous two-phase PEG/dextran systems. With less than 1% of the PEG substituted with chelated copper, partition coefficients are shown to increase by factors of up to 37 over extraction with unsubstituted PEG. The proteins studied are preferentially extracted into the Cu(II)PEGIDA phase in proportion to the number of accessible histidine residues on their surface. The affinity contribution to partitioning is proportional to the number of exposed histidine over a very wide range. The partition coefficients of heme-containing proteins measured in the Cu(II)PEG-IDA/dextran systems increase with the pH of the extraction mixture from pH 5.5 to pH 8.0, while partition coefficients in the unsubstituted PEG/dextran systems are very nearly independent of pH. The strong pH dependence of the metalaffinity extraction can be utilized in the recovery of the extracted protein.     

Overproduction of single-chain antibodies against human IFN-alpha2B in Escherichia coli and their isolation in biologically active form

The gene encoding mouse single chain antibody (ScFv) against human interferon alpha2b (IFN-alpha2b) was cloned into the plasmid vector under the control of promoter from phage T7 and the recombinant protein was expressed in Escherichia coli as inclusion bodies. After the isolation of inclusion bodies the desired protein containing affinity tail "6His tag" was solubilized and purified under denaturing conditions by immobilized-metal affinity chromatography. The soluble and purified ScFv was obtained by "on column" refolding and the recovery of biological activity were demonstrated. The higher levels of ScFv production for intracellular expression system in comparison with ScFv obtained by secretion were shown. The advantages of described refolding method are simplicity and high efficacy, moreover, refolding using a chromatographic process represents the manufacturable approach because it is easily automated using commercially available materials and preparative chromatography systems and also can be combined with simultaneous purification.     

The affinity concept in bioseparation: evolving paradigms and expanding range of applications

The meaning of the word affinity in the context of protein separation has undergone evolutionary changes over the years. The exploitation of molecular recognition phenomenon is no longer limited to affinity chromatography modes. Affinity based separations today include precipitation, membrane based purification and two-phase/three-phase extractions. Apart from the affinity ligands, which have biological relationship (in vivo) with the target protein, a variety of other ligands are now used in the affinity based separations. These include dyes, chelated metal ions, peptides obtained by phage display technology, combinatorial synthesis, ribosome display methods and by systematic evolution of ligands by exponential enrichment (SELEX). Molecular modeling techniques have also facilitated the designing of biomimetic ligands. Fusion proteins obtained by recombinatorial methods have emerged as a powerful approach in bioseparation. Overexpression in E. coli often result in inactive and insoluble inclusion bodies. A number of interesting approaches are used for simultaneous refolding and purification in such cases. Proteomics also needs affinity chromatography to reduce the complexity of the system before analysis by electrophoresis and mass spectrometry are made. At industrial level, validation, biosafety and process hygiene are also important aspects. This overview looks at these evolving paradigms and various strategies which utilize affinity phenomenon for protein separations.     

Surface topography of histidine residues in lysozymes.

Several avian and mammalian c-type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA-M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal-ion affinity chromatography.     

Metal affinity extraction of human hemoglobin in an aqueous polyethylene glycol-sodium sulfate two-phase system

Summary We have developed a protein extraction technique which uses metal affinity ligands in PEG/salt aqueous two-phase systems. Cu(II)IDA-PEG will partition proteins according to their surface histidine contents in two-phase systems formed from sodium sulfate and polyethylene glycol. The nearly complete separation of human hemoglobin and human serum albumin in a single stage is presented as a demonstration of the effectiveness of metal affinity extraction in PEG/salt systems.     

High recovery refolding of rhG-CSF from Escherichia coli, using urea gradient size exclusion chromatography

Protein folding liquid chromatography (PFLC) is a powerful tool for simultaneous refolding and purification of recombinant proteins in inclusion bodies. Urea gradient size exclusion chromatography (SEC) is a recently developed protein refolding method based on the SEC refolding principle. In the presented work, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli (E. coli) in the form of inclusion bodies was refolded with high yields by this method. Denatured/reduced rhG-CSF in 8.0 mol.L(-1) urea was directly injected into a Superdex 75 column, and with the running of the linear urea concentration program, urea concentration in the mobile phase and around the denatured rhG-CSF molecules was decreased linearly, and the denatured rhG-CSF was gradually refolded into its native state. Aggregates were greatly suppressed and rhG-CSF was also partially purified during the refolding process. Effects of the length and the final urea concentration of the urea gradient on the refolding yield of rhG-CSF by using urea gradient SEC were investigated respectively. Compared with dilution refolding and normal SEC with a fixed urea concentration in the mobile phase, urea gradient SEC was more efficient for rhG-CSF refolding--in terms of specific bioactivity and mass recovery, the denatured rhG-CSF could be refolded at a larger loading volume, and the aggregates could be suppressed more efficiently. When 500 microL of solubilized and denatured rhG-CSF in 8.0 mol.L(-1) urea solution with a total protein concentration of 2.3 mg.mL(-1) was loaded onto the SEC column, rhG-CSF with a specific bioactivity of 1.0 x 10(8) IU.mg(-1) was obtained, and the mass recovery was 46.1%.     

His‐tagged protein purification by metal‐chelate affinity extraction with nickel‐chelate reverse micelles

Di(2‐ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X‐45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R₂. The Ni(II)‐chelate reverse micelles were characterized for the purification of recombinant hexahistidine‐tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R₂ was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni²⁺ ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two‐stage purification by the metal‐chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments

Immobilized metal-ion affinity chromatography (IMAC) performed with four different transition metal ions: copper(II), nickel(II), zinc(II) and cobalt(II), was used to study the adsorption properties of human polyclonal gamma-globulines (IgG), Cohn II-III fractions, and their pepsin cleaved fragments: Fab'2 and F'c. In each case, digested products showed lower affinity for metal ions, as well by decreasing pH elution as by competition with imidazole. An explanation was proposed by the presence of a histidine (His) cluster in the F'c domain of IgGs, identified by computer calculation (accessible surface area (ASA) determination) as the more probable His 433-x-His 435 sequence presented in the CH3 domain of human IgG heavy chain. As shown by IMAC and electrophoresis, F'c and undigested IgG have higher affinity for transition metal ions than Fab'2 fragments and could be then separated in one step by IMAC. When chelated Zn(II) or Co(II) are used as ligands, the Fab'2 fragment could be easily recovered under mild conditions (pH 7) in the non-retained fraction. This approach could be used as a powerful alternative to conventional protein A/G methods for the commercial preparation of non immunogen active Fab'2 fragments.     

Non-chromatographic strategies for protein refolding

Overexpression of recombinant proteins in bacterial systems (such as E. coli) often leads to formation of inactive and insoluble ' inclusion bodies' . Protein refolding refers to folding back the proteins after solubilizing/unfolding the misfolded proteins of the inclusion bodies. Protein aggregation, a concentration dependent phenomenon, competes with refolding pathway. The refolding strategies largely aim at reducing aggregation and/or promoting correct folding. This review focuses on non-chromatographic strategies for refolding like dilution, precipitation, three phase partitioning and macro-(affinity ligand) facilitated three phase partitioning. The nanomaterials which disperse well in aqueous buffers are also discussed in the context of facilitating protein refolding. Apart from general results with these methods, the review also covers the use of non-chromatographic methods in protein refolding in the patented literature beyond 2000. The patented literature generally describes use of cocktail of additives which results in increase in refolding yield. Such additives include low concentration of chaotropic agents, redox systems, ions like SO4(2-) and Cl-, amines, carboxylic acids and surfactants. Some novel approaches like use of a "pressure window" or ionic liquids for refolding and immobilized diselenide compounds for ensuring correct -S-S- bonds pairing have also been discussed in various patents. In most of the patented literature, focus naturally has been on refolding in case of pharmaceutical proteins.     

Transition metal complexes of terminally protected peptides containing histidyl residues

Histidine-containing peptide fragments of prion protein are efficient ligands to bind various transition metal ions and they have high selectivity in metal binding. The metal ion affinity follows the order: Pd(II)>Cu(II)>Ni(II)Zn(II)>Cd(II) approximately Co(II)>Mn(II). The high selectivity of metal binding is connected to the involvement of both imidazole and amide nitrogen atoms in metal binding for Pd(II), Cu(II) and Ni(II), while only the monodentate N(im)-coordination is possible with the other metal ions. The stoichiometry and binding mode of palladium(II) complexes show great variety depending on the metal ion to ligand ratio, pH and especially the presence of coordinating donor atoms in the side chains of peptide fragments. It is also clear from our data that the peptide fragments containing histidine outside the octarepeat (His96, His111 and His187) are more efficient ligands than the monomer peptide fragments of the octarepeat domain.     

Comparison of classical and affinity purification techniques of Mason-Pfizer monkey virus capsid protein: the alteration of the product by an affinity tag

The efficiencies of different procedures for purification of the capsid protein (CA) of Mason-Pfizer monkey virus are compared. Plasmids encoding both wild-type CA and two C-terminally modified sequences of CA suitable for affinity chromatography purification were prepared. CA was expressed in Escherichia coli (i) as a wild-type protein, (ii) C-terminally extended with a six-histidine tag (CA 6His), and (iii) as a protein containing a C-terminal fusion to a viral protease cleavage site followed by a six-histidine tag (CA 6aa6His). Electron microscopy was used for comparison of the resulting proteins, as CA is a structural protein with no enzymatic activity. We have found that these C-terminal fusions dramatically influenced the properties and morphology of structures formed by CA protein in E. coli. The formation of amorphous aggregates of CA was abolished and CA 6His and CA 6aa6His proteins formed organized structures. CA and CA 6aa6His accumulated in bacteria in inclusion bodies as insoluble proteins, CA 6His was found in a soluble form. Both six-histidine-tagged proteins were purified using affinity chromatography under either native (CA 6His) or denaturing (CA 6aa6His) conditions. CA protein was purified under denaturing conditions using gel-filtration chromatography followed by refolding. All proteins were obtained at a purity >98%. Both aforementioned C-terminal extensions led to dramatic changes in behavior of the products and they also affected the tendency to form organized structures within E. coli. We show here that the widely used histidine anchor may significantly alter the properties of the protein of interest.     

Expression, single-step purification, and matrix-assisted refolding of a maize cytokinin glucoside-specific beta-glucosidase.

Availability of highly purified native beta-glucosidase Zm-p60.1 in milligram quantities was a basic requirement for analysis of structure-function relationships of the protein. Therefore, Zm-p60.1 was overexpressed to high levels as a fusion protein with a hexahistidine tag, (His)(6)Zm-p60.r, in Escherichia coli, resulting, however, in accumulation of most of the protein in insoluble inclusion bodies. Native (His)(6)Zm-p60.r was then purified either from the bacterial lysate soluble fraction or from inclusion bodies. In the first case, a single-step purification under native conditions based on immobilized metal affinity chromatography (IMAC) was developed. In the second case, a single-step purification protocol under denaturing conditions followed by IMAC-based matrix-assisted refolding was elaborated. The efficiency of the native protein purification from soluble fraction of bacterial homogenate was compared to the feasibility of purification and renaturation of the protein from inclusion bodies. Gain of authentic biological activity and quaternary structure after the refolding process was confirmed by K(m) determination and electrophoretic mobility under native conditions. The yield of properly refolded protein was assessed based on the specific activity of the refolded product.     

Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein

The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60–91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91–230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).     

Purification of recombinant antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein antigen using the expression system pH6EX3 followed by metal chelating affinity chromatography.

A novel plasmid expression vector (pH6EX3) that directs the synthesis of a fusion protein with a histidine hexapeptide at its N-terminus and a foreign protein at its C-terminus was constructed. The fusion gene is controlled by a strong tac promoter, leading to high-level expression of recombinant protein in several bacterial strains; the protein is deposited mainly as an insoluble mass in inclusion bodies. The fusion protein can be purified from the insoluble cell fraction by one-step affinity chromatography based on the selective interaction between the histidine hexapeptide and a metal chelating matrix charged with Ni2+ ions. The principle of this new system was tested by expressing and purifying antigenic epitopes of the human 68-kDa (U1) ribonucleoprotein autoantigen. With the use of column chromatography and pH gradient elution, about 25 micrograms recombinant protein/ml of bacterial culture was obtained.     

Intensified process for the purification of an enzyme from inclusion bodies using integrated expanded bed adsorption and refolding

This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations in an intensified process used to recover purified and biologically active proteins from inclusion bodies expressed in E. coli. Delta(5)-3-Ketosteroid isomerase with a C-terminal hexahistidine tag was expressed as inclusion bodies in the cytoplasm of E. coli. Chemical extraction was used to disrupt the host cells and simultaneously solubilize the inclusion bodies, after which EBA utilizing immobilized metal affinity interactions was used to purify the polyhistidine-tagged protein. Adsorptive refolding was then initiated in the column by changing the denaturant concentration in the feed stream from 8 to 0 M urea. Three strategies were tested for performing the refolding step in the EBA column: (i) the denaturant was removed using a step change in feed-buffer composition, (ii) the denaturant was gradually removed using a gradient change in feed-buffer composition, and (iii) the liquid flow direction through the column was reversed and adsorptive refolding performed in the packed bed. Buoyancy-induced mixing disrupted the operation of the expanded bed when adsorptive refolding was performed using either a step change or a rapid gradient change in feed-buffer composition. A shallow gradient reduction in denaturant concentration of the feed stream over 30 min maintained the stability of the expanded bed during adsorptive refolding. In a separate experiment, buoyancy-induced mixing was completely avoided by performing refolding in a settled bed, which achieved comparable yields to refolding in an expanded bed but required a slightly more complex process. A total of 10% of the available KSI-(His(6)) was recovered as biologically active and purified protein using the described purification and refolding process, and the yield was further increased to 19% by performing a second iteration of the on-column refolding operation. This process should be applicable for other polyhistidine tagged proteins and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution.     

Protein separation using metal ion-bound particles in aqueous two-phase system

Metal ion affinity partitioning of protein in aqueous two-phase systems was studied using Sepharose as ligand carrier as an integrated adsorption partitioning. Cu(II)-bound Sepharose was mixed with protein solution and an aqueous two-phase system. The affinity sorbent was distributed quantitatively to the upper side or the interface. The binding studies of lysozyme to copper-bound gel in PEG/dextran two-phase systems demonstrate the feasibility of this bioseparation process. PEG/dextran system did not affect binding and elution of lysozyme to and from the Cu(II)-Sepharose particles.     

One-step Purification and Immobilization in Cellulose of the GroEL Apical Domain Fused to a Carbohydrate-binding Module and its use in Protein Refolding

The apical domain of the chaperonin, GroEL, fused to the carbohydrate binding module type II, CBD_Cex, of Cellulomonas fimi, was expressed in Escherichia coli. The recombinant protein, soluble or from inclusion bodies, was directly purified and immobilized in microcrystalline cellulose particles or cellulose fabric membranes. Assisted refolding of rhodanese by the immobilized mini-chaperone showed a two-fold improvement as compared to a control. Using chromatographic refolding, 35% of rhodanese activity was recovered in only 5 min (mean residence time) as compared to 17% for spontaneous refolding. This mini-chaperone immobilized in cellulose could be a cost-efficient method to refold recombinant proteins expressed as inclusion bodies.     

Purification of histidine-tagged single-chain Fv-antibody fragments by metal chelate affinity precipitation using thermoresponsive copolymers

Metal chelate affinity precipitation (MCAP) has been successfully developed as a simple purification process for proteins that have affinity for metal ions. The present lack of widespread applications for this technique as compared to immobilized metal affinity chromatography (IMAC) may be related to the scarcity of well-characterized metal affinity macroligands (AML) and their applications to the number of different purification systems. In the present work we describe a detailed study of a new purification system using metal-loaded thermoresponsive copolymers as AML. The copolymers of vinylimidazole (VI) with N-isopropylacrylamide (NIPAM) were synthesized by radical polymerization with imidazole contents of 15 and 24 mol%. When loaded with Cu(II) and Ni(II) ions the copolymers selectively precipitated extracellularly expressed histidine-tagged single-chain Fv-antibody fragments (His(6)-scFv fragments) from the fermentation broth free from E. coli cells. Precipitation was induced by salt at mild temperatures and the bound antibody fragments were recovered by dissolving the protein-polymer complex in EDTA buffer and subsequent reprecipitation of the polymer. His(6)-scFv fragments were purified with yields of 91 and 80% and purification folds of 16 and 21 when Cu(II) and Ni(II) copolymers were used, respectively. The protein precipitation capacity of the Ni(II) copolymer showed a dependence on the VI concentration in the copolymer. The SDS-PAGE pattern showed significant purification of the antibody fragments.     

Affinity extraction of dye- and metal ion-binding proteins in polyvinylpyrrolidone-based aqueous two-phase system

Affinity extraction of dye- and metal ion-binding proteins, respectively, in a polyvinylpyrrolidone (PVP40)-Reppal PES 100 two-phase system was investigated. Due to the ability of PVP to complex azo dyes and inorganic ions, covalent coupling of the ligands was not essential. Cibacron Blue F3GA was used as the ligand for extraction of lactate dehydrogenase (LDH) from porcine muscle, while copper ions were used for extraction of B. stearothermophilus LDH with a fusion tag of six histidine residues (His6-LDH) from recombinant Escherichia coli homogenate. The binding strength of the enzymes to their respective ligands was only slightly reduced in the presence of PVP. The partition coefficient of Cibacron Blue and Cu2+ ions in the two-phase systems composed of different concentrations of PVP and Reppal was in the range of 20-30, with maximal partitioning being observed in the 17% (w/w) PVP40-10% Reppal PES100 system. Only a minor leakage of the ligands to the bottom phase was observed with time. The partitioning of porcine LDH to the PVP phase was increased 100-fold, and a maximal recovery of 89% was obtained in the two-phase system loaded with 0.2% (w/w) Cibacron Blue. The enzyme was quantitatively recovered with further purification from the PVP-dye phase using a secondary extraction step with 170 mM phosphate or alternatively with 100 mM phosphate containing NADH or NaCl. A more than 10-fold increase in the partition coefficient of His6-LDH was achieved in the two-phase system loaded with 0.4% (w/w) copper sulfate compared to the system lacking the metal ions. The enzyme was also back-extracted into phosphate phase in the presence of imidazole.