Perspectives of immobilized-metal affinity chromatography.

Immobilized Metal-Affinity Chromatography (IMAC) represents a relatively new separation technique that is primarily appropriate for the purification of proteins with natural surface-exposed histidine residues and for recombinant proteins with engineered histidine tags or histidine clusters. Because the method has gained broad popularity in recent years, the main recent developments in the field of new sorbents, techniques and possible applications are discussed in this article. Advantages of the method and new prospects are described as well as the problems and concerns that appear when the method is to be used for production of pharmaceutical-grade proteins.     

Characteristics and applications of adsorbents for pyrogen removal

Characteristics and applications of immobilized histidine and immobilized histamine for pyrogen removal were investigated. Immobilized histidine showed a high affinity for pyrogen at low ionic strength and over a wide pH range. The adsorption capacity was 0.53 mg of lipopolysaccharide per milliliter of the adsorbent. The apparent dissociation constant was 1.57 X 10(-9) M. The adsorption of pyrogen to immobilized histidine decreased with increasing ionic strength, but pyrogen could be adsorbed even at ionic strengths of gamma/2 = 0.05-0.1, at which other substances were little adsorbed; that is, specific adsorption of pyrogen was observed. The adsorption of pyrogen could be increased at ionic strengths of gamma/2 = 0.05-0.1 by using a lower flow rate or a longer column length. Immobilized histidine and immobilized histamine could be used for the removal of natural pyrogens contaminating various useful low-molecular-weight compounds as well as high-molecular-weight compounds such as proteins.     

Affinity purification of histidine-tagged proteins

Expression of recombinant proteins is a standard technique in molecular biology and a wide variety of prokaryotic as well as eukaryotic expression systems are currently in use. A limiting step is often the purification of the expressed recombinant protein, particularly if mammalian expression systems that yield low expression levels are employed. Here, we discuss the advantages and restrictions of tagging recombinant proteins with histidines and purifying them by Ni²⁺-NTA chromatography.Abbreviations GST glutathione S-transferase - NTA nitrilotriacetic acid - His histidine - PAGE polyacrylamide gel electrophoresis     

Immunoaffinity chromatography

The basic procedure of immunoaffinity chromatography (IAC) is described. The insoluble support matrices available for IAC and their activation chemistries, including some of the most recently introduced, are reviewed. Means of selecting the most appropriate monoclonal antibody (MAb) are described, although an empirical approach is still required for the final choice of antibody. Precise methods of runing IAC columns are surveyed including the binding, washing, and elution stages, although no precise recommendations can be made particularly for the elution step since this is unique to a particular MAb and antigen. All IAC sorbents lose activity with time through a combination of MAb inactivation and ligand leakage. The relative importance of the two phenomena is discussed, and suggestions are made to minimize the problem along with an indication of the relative stabilities of a range of coupling chemistries. A sample of the proteins purified by IAC is given together with pointers to the future of the technique.     

Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags

The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.     

Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity. (c) 1996 John Wiley & Sons, Inc.     

Specific removal of endotoxin from protein solutions by immobilized histidine

A method for reducing endotoxin contamination in various solutions by immobilized histidine is described. Immobilized histidine is a porous adsorbent suitable for the adsorption of endotoxin with a high affinity over a wide range of pH and temperature and at low ionic strength (gamma/2 less than or equal to 0.1). When a purified endotoxin originating from Escherichia coli UKT-B was studied, the apparent dissociation constant between endotoxin and the adsorbent was 7.3 X 10(-13) M. The adsorbent was able to remove various kinds of endotoxin originating from gram-negative bacteria; the concentration of endotoxin was reduced from 1000 to less than 0.01 ng/ml in water. It is shown that the adsorbent specifically adsorbs endotoxin provided that the adsorption conditions are properly selected. Some examples of the specific removal of endotoxin from high-molecular-weight physiologically active substances such as tumor necrosis factor and lysozyme are shown.     

The association of myelin basic protein with itself and other proteins

Chromatographic studies were performed to measure myelin basic protein (MBP) interactions by covalently binding a number of different proteins to Sepharose and passing radioactive bovine MBP over these columns. Studies at a variety of pH values, ionic strengths and temperatures revealed that the bovine MBP could interact with itself as well as cytochrome c, lysozyme, and ovalbumin. Chromatographic profiles of elution volume vs. pH revealed that the interaction between MBP and these immobilized proteins was biphasic. The self-association of MBP was found to be strongest between pH 7.4 and 8.1 and at an elevated temperature. Titration of the amino acid residues responsible for the association of MBP with other proteins revealed apparent pKs ranging from 6.10 to 6.70. A pH dependence study at an elevated temperature shifted the apparent pK of the MBP interaction to a lower value with all the proteins except ovalbumin. After destroying 60% of the histidine residues in MBP by photooxidation and passing¹²⁵I-labeled photooxidized MBP over Sepharose columns containing immobilized protein, the second phase in binding was decreased significantly with immobilized cytochrome c, lysozyme, and MBP and to a smaller extent with ovalbumin. These results are consistent with the involvement of deprotonated histidine residues in the MBP-protein associations.     

A heme fusion tag for protein affinity purification and quantification

We report a novel affinity‐based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an L ‐histidine‐immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme‐tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag‐HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination.     

Affinity chromatography of nucleases

The review concerns isolation and purification of nucleases by affinity chromatography. Different stationary ligands and the methods for their immobilization on supports are described, along with diverse eluents and various procedures for a nuclease detachment from the affinity sorbents. The data on the affinity chromatography application for measuring the dissociation constants of the enzyme complexes with either immobilized or soluble ligands are compiled.     

Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

Thermally responsive elastin like polypeptides (ELPs) can be used to purify proteins from Escherichia coli culture when proteins are expressed as a fusion with an ELP. Nonchromatographic purification of ELP fusion proteins, termed inverse transition cycling (ITC), exploits the reversible soluble-insoluble phase transition behavior imparted by the ELP tag. Here, we quantitatively compare the expression and purification of ELP and oligohistidine fusions of chloramphenicol acetyltransferase (CAT), blue fluorescent protein (BFP), thioredoxin (Trx), and calmodulin (CalM) from both a 4-h culture with chemical induction of the plasmid-borne fusion protein gene and a 24-h culture without chemical induction. The total protein content and functional activity were quantified at each ITC purification step. For CAT, BFP, and Trx, the 24-h noninduction culture of ELP fusion proteins results in a sevenfold increase in the yield of each fusion protein compared to that obtained by the 4-h-induced culture, and the calculated target protein yield is similar to that of their equivalent oligohistidine fusion. For these proteins, ITC purification of fusion proteins also results in approximately 75% recovery of active fusion protein, similar to affinity chromatography. Compared to chromatographic purification, however, ITC is inexpensive, requires no specialized equipment or reagents, and because ITC is a batch purification process, it is easily scaled up to accommodate larger culture volumes or scaled down and multiplexed for high-throughput, microscale purification; thus, potentially impacting both high-throughput protein expression and purification for proteomics and large scale, cost-effective industrial bioprocessing of pharmaceutically relevant proteins.     

A new chromatographic method for the preparative isolation of phosphoinositides and other anionic phospholipids

A new chromatographic procedure for preparative isolation of mono-, di- and triphosphoinositides and other anionic phospholipids with the use of adsorbents containing primary amino groups is described. Sorbents with immobilized neomycin, L-lysine and aminoalkyl groups were tested. Conditions for isolation of chromatographically pure phospholipids of separate classes on the above sorbents were developed. Isolation of polyphosphoinositides on the amino sorbents represents a new type of chromatography involving bioaffinity and ion-exchange interaction.     

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

蛋白组氨酸磷酸酶研究进展

主要概括磷酸酶的种类,原核细胞磷酸组氨酸生物功能及调控,哺乳动物组氨酸残基磷酸化、去磷酸化,以及组氨酸磷酸酶及其底物的最新研究进展. 信号转导在生长发育及细胞功能中起极其重要的作用. 无论在原核还是真核细胞,蛋白质磷酸化是细胞内信号转导的关键机制. 研究最多的可逆的真核蛋白磷酸化,主要发生在含有羟基的丝氨酸、苏氨酸和酪氨酸残基上. 不同的激酶和磷酸酶受不同机制的调节,而调节过程中出现的差异是人类很多疾病的潜在基础. 与大量有关羟基磷酸化氨基酸的报道相比,有关氨基磷酸化氨基酸的报道甚少. 据估计,自然界中存在的磷酸组氨酸比磷酸酪氨酸多10 ~ 100倍,但不如磷酸丝氨酸丰富. 虽然对脊椎动物蛋白质中存在磷酸组氨酸的认识可以追溯到20世纪60年代初, 但由于研究手段的限制,至今对脊椎动物蛋白组氨酸激酶及组氨酸磷酸酶的结构及功能知之甚少. 但是,近几年的研究有突破性的发现,克隆和重组表达哺乳动物组氨酸磷酸酶为研究氨基磷酸化氨基酸的生物功能翻开新的一章.     

Tandem affinity monolithic microcolumns with immobilized protein A, protein G', and antibodies for depletion of high abundance proteins from serum samples: integrated microcolumn-based fluidic system for simultaneous depletion and tryptic digestion

Affinity monolithic microcolumns with immobilized affinity ligands including protein A, protein G' and polyclonal antibodies were developed for the microscale depletion of the top eight most abundant proteins in human serum. These various affinity microcolumns were evaluated for their sample loading capacities with the standard protein substrates. In general, the sample loading capacity of protein A and protein G' was about 7-25 fold higher than that of the antibody-based affinity columns. The macroporous nature of the monolithic columns, which offers high permeability in pressure-driven flow, allowed the design of long tandem affinity columns for the simultaneous depletion of the top eight most abundant proteins in a single run. The tandem format could be extended to include additional affinity monolithic columns to deplete other proteins for which specific antibodies are available without running into high inlet pressure. Furthermore, the tandem affinity columns were integrated with immobilized trypsin monolithic columns to achieve the simultaneous depletion and digestion of proteins. The various formats investigated in this study could be down scaled to achieve nanoLC or up scaled to perform conventional HPLC depending on the size of the proteomic samples.     

Separation of hexahistidine fusion proteins with immobilized metal ion affinity chromatographic (IMAC) sorbents derived from MN+‐tacn and its derivatives

The capabilities of a new class of immobilized (im) metal ion chelate complexes (IMCCs), derived from 1,4,7‐triazacyclononane (tacn), bis(1,4,7‐triazacyclononyl) ethane (dtne) and bis(1,4,7‐triazacyclononyl)propane (dtnp) complexed with the borderline metal ions Cu²⁺, Ni²⁺, Zn²⁺, Mn²⁺, Co²⁺, and Cr³⁺, for the purification of proteins have been investigated. In particular, the binding behavior of a model protein, the C‐terminal hexahistidine tagged recombinant fusion protein Schistosoma japonicum glutathione S‐transferase‐Saccharomyces cerevisiae mitochondrial ATP synthase δ‐subunit (GST‐δATPase‐His₆), with these new immobilized metal ion affinity chromatographic (IMAC) sorbents was compared to the properties of a conventional sorbent, derived from immobilized Ni(II)‐nitrilotriacetic acid (im‐Ni²⁺‐NTA). Investigations using the recombinant GST‐δATPase‐His₆ and recombinant S. japonicum glutathione S‐transferase (GST) lacking a hexahistidine tag have confirmed that the C‐terminal tag hexahistidine residues were required for the binding process to occur with these IMAC systems. The results also confirm that recombinant fusion proteins such as GST‐δATPase‐His₆ can be isolated in high purity with these IMAC systems. Moreover, these new macrocyclic systems manifest different selectivity features as a function of pH or ionic strength when compared to the conventional, unconstrained iminodiacetic acid (IDA) or NTA chelating ligands, complexed with borderline metal ions such as Cu²⁺ or Ni²⁺, as IMAC systems. Biotechnol. Bioeng. 2009;103: 747–756. © 2009 Wiley Periodicals, Inc.     

Expression and simple,one-step purification of fragile histidine triad (Fhit) tumor suppressor mutant forms in <Emphasis Type="Italic">Escherichia coli</Emphasis> and their interaction with protoporphyrin IX

The product of human fragile histidine triad (FHIT) gene is a tumor suppressor protein of still largely unknown cellular background. We have shown previously that it binds protoporphyrin IX (a photosensitizer) which alters its enzymatic activity in vitro. Fhit, diadenosine triphosphate (Ap₃A) hydrolase, possesses the active site with histidine triad His-φ-His-φ-His-φφ. So-called histidine Fhit mutants (His94Asn, His96Asn and His98Asn) exhibit highly reduced activity in vitro, however, their antitumor function has not been fully described yet. In this work we have cloned the cDNAs of histidine mutants into pPROEX-1 vector allowing the production of His₆-fusion proteins. The mutated proteins: Fhit-H94N, Fhit-H96N and Fhit-H98N, were expressed in Escherichia coli BL21(DE3) and purified (up to 95%) by an improved, one-step affinity chromatography on Ni-nitrilotriacetate resin. The final yield was 2 mg homogenous proteins from 1 g bacteria (wet wt). The activity of purified proteins was assessed by previously described assay. The same purification procedure yielded 0.8 mg/ml and highly active wild-type Fhit protein (K _m value for Ap₃A of 5.7 μM). Importantly, purified mutant forms of Fhit also interact with a photosensitizer, protoporphyrin IX in vitro.     

Engineered protein a for the orientational control of immobilized proteins

This work describes the genetic engineering and characterization of a histidine-tagged fragment of protein A. The histidine tag results in the site-selective immobilization of the protein A receptor and the preservation of its high ligand affinity when immobilized on solid supports. The fragment was expressed at high yield in E. coli and purified to homogeneity. When selectively immobilized to histidine binding matrices, the protein A fragment exhibits high affinity for soluble IgG. We further demonstrate from adsorption isotherms that the receptor exhibits a homogeneous, high affinity population at densities where steric crowding between large ligands does not affect the apparent receptor affinity. This engineered receptor is appropriate for a range of applications including sensor design or those using immobilized Fc-tagged proteins.     

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

Process intensification for the removal of poly‐histidine fusion tags from recombinant proteins by an exopeptidase

This study describes the use of a hexa‐histidine tagged exopeptidase for the cleavage of hexa‐histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On‐column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on‐column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post‐load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post‐load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa‐histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa‐histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on‐column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on‐column exopeptidase cleavage makes the integration of the poly‐histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly‐histidine tag removal can now be combined with the on‐column tag cleavage operation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010