Hexahistidine-tagged Recombinant Protein Research Articles

An ssDNA aptamer specific for detection and purification of hexahistidine-tagged proteins

Aptamers are small-sized RNA or ssDNA ligands with a unique structure, which have high specificity and affinity to their cognate targets. Thus, in addition to the extensive values in various bio-medical fields, aptamers can also be alternatively used as affinity ligands in the bioprocess, such as for protein purification. In the present study, a hexahistidine specific aptamer named AptHis-C, was developed through the SELEX methodology, which has high affinity to hexahistidine, and its dissociation constant was as low as 20.8 nM. The structural prediction revealed that AptHis-C contains two connected stem-loop conformations. AptHis-C can only specifically recognize recombinant proteins with the hexahistidine-tag in simple or complex situations, and not to those with other tags. When immobilized on magnetic beads, AptHis-C can be used as a tool for hexahistidine-tagged recombinant protein purification. Its effectiveness is as good as traditional Ni-based beads. Besides, due to the intrinsic characteristics of nucleic acids, such as high thermal/chemical stability, immobilized aptamer-magnetic beads can be reused many times without an obvious decrease of purification effectiveness. This aptamer may represent a novel method for the detection and purification of hexahistidine-tagged recombinant proteins.

Selective Derivatization of Hexahistidine-Tagged Recombinant Proteins.

Covalent modification of proteins is extensively used in research and industry for biosensing, medical diagnostics, targeted drug delivery, and many other practical applications. The conventional method for production of protein conjugates has changed little in the last 20years mostly relying on reactions of side chains of cysteine and lysine residues. Due to the presence of large numbers of similar reactive amino acid residues in proteins, common synthetic methods generally produce complex mixtures of products, which are difficult to separate. An emerging alternative technology for covalent modification of proteins involves formation of a covalent bond with a hexahistidine affinity tag present in a majority of recombinant proteins without interfering with other amino acid residues. The approach is based on formation of a ternary complex of the hexahistidine sequence with a bivalent metal cation chelated by ligand bearing an electrophilic Baylis-Hillman ester group capable of subsequent formation of a covalent bond with one of the histidine residues of the tag. The reaction proceeds under mild reaction conditions in neutral aqueous solutions under high dilutions (10-5 to 10-4M) providing a stable covalent bond between the label and an imidazole residue in a hexahistidine tag at either C- or N-terminus. Because hexahistidine affinity tag methodology is a de-facto standard for preparation of recombinant proteins our approach can be easily implemented for selective derivatization of these proteins with fluorescent groups, alkynyl groups for "click" reactions, or biotinylation.

Improving the production of the denatured recombinant N-terminal domain of rhoptry-associated protein 2 from a Plasmodium falciparum target in the pathology of anemia in falciparum malaria

Rhoptry-associated protein 2 (RAP2) is known to be discharged from rhoptry onto the membrane surface of infected and uninfected erythrocytes (UEs) ex vivo and in vitro and this information provides new insights into the understanding of the pathology of severe anemia in falciparum malaria. In this study, a hexahistidine-tagged recombinant protein corresponding to residues 5-190 of the N-terminal of Plasmodium falciparum RAP2 (rN-RAP2) was produced using a new method of solubilization and purification. Expression was induced with D-lactose, a less expensive alternative inducer to the more common isopropyl-beta-D-thio-galactopyranosidase. The recombinant protein was purified using two types of commercially-available affinity columns, iminodiacetic and nitrilotriacetic. rN-RAP2 had immunogenic potential, since it induced high titers of anti-RAP2 antibodies in mice. These antibodies recognized full-length RAP2 prepared from Triton X-100 extracts from two strains of P. falciparum. In fact, the antibody recognized a 29-kDa product of RAP2 cleavage as well as 82 and 70-kDa products of RAP1 cleavage. These results indicate that the two antigens share sequence epitopes. Our expressed protein fragment was shown to contain a functional epitope that is also present in rhoptry-derived ring surface protein 2 which attaches to the surface of both infected and UEs and erythroid precursor cells in the bone marrow of malaria patients. Serum from malaria patients who developed anemia during infection recognized rN-RAP2, suggesting that this protein fragment may be important for epidemiological studies investigating whether immune responses to RAP2 exacerbate hemolysis in falciparum malaria patients.

Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design

Its natural resistance to antiprotozoal chemotherapy characterizes the intestinal protozoan parasite Cryptosporidium parvum and the P-glycoprotein-related multidrug resistance proteins such as CpABC3 could be involved. In order to design and study specific inhibitors of the CpABC3 nucleotide-binding domain, a hexahistidine-tagged recombinant protein encompassing the N-terminal cytosolic NBD1 domain was overexpressed in E. coli and purified. The 45kDa H6-NBD1 displayed intrinsic fluorescent properties consistent with the presence of two Trp residues in a hydrophobic environment. The binding of ATP and the fluorescent analogue TNP-ATP produced a dose-dependent quenching as well as progesterone and the flavone quercetin. The extrinsic fluorescence of TNP-ATP was enhanced upon binding to H6-NBD1, which was only partially displaced by the natural substrate ATP. The recombinant protein hydrolyzed ATP (Km=145.4±18.2μM), but ADP (Km=4.3±0.6mM) and AMP (Km=5.4±1.5μM) were also substrates. TNP-ATP is a competitive inhibitor of the catalytic activity (Ki=36.6±4.5μM), but quercetin and progesterone were not inhibitors, evidencing different binding sites. The recombinant C. parvum H6-NBD1 should be a valuable tool for rational drug design and will allow the discrimination between specific inhibitors of the catalytic site and molecules binding to other sites.

Molecular cloning and functional expression of rabbit α2-antiplasmin

The rabbit is frequently employed in small animal models of in-vivo coagulation and fibrinolysis, but the degree to which its plasma proteins resemble their human counterparts is incompletely known. Our aims were: to determine the nucleotide sequence of a full-length rabbit liver alpha(2)-antiplasmin (alpha(2)AP) cDNA; compare it with its human, murine, and bovine counterparts; and express it in functional form. Partial cDNAs encoding 5' and 3' portions of the alpha(2)AP mRNA were obtained by reverse transcriptase (RT)-polymerase chain reaction (PCR), using rabbit liver RNA and 'guessmer' oligonucleotides based on known sequences. This information was employed to design additional oligonucleotides for RT-PCR and rapid amplification of cDNA ends (RACE) procedures yielding overlapping clones corresponding to the entire rabbit alpha(2)AP mRNA sequence. Mature alpha(2)AP was expressed as a hexahistidine-tagged recombinant protein in Escherichia coli, purified by nickel-chelate affinity and ion exchange chromatography, and its reaction with plasmin and soluble fibrin assessed electrophoretically and compared with an analogous recombinant human alpha(2)AP. The consensus rabbit alpha(2)AP cDNA is 2,157 bp long, and encodes a 464-residue mature sequence and a 27-amino-acid presequence. The mature protein is 82% identical to its human counterpart, and its recombinant form produced denaturation-resistant complexes with both plasmin and fibrin. The region of greatest sequence divergence lies within the C-terminal extension of alpha(2)AP, unique in the serpin family of proteins to which alpha(2)AP belongs. The highly conserved nature of rabbit alpha(2)AP reinforces its role as a vital antifibrinolytic protein and supports fibrinolytic investigations in models employing this small animal.

Structure of glyceraldehyde-3-phosphate dehydrogenase fromPlasmodium falciparum

The malaria parasite Plasmodium falciparum is responsible for about two million deaths annually, making it important to obtain information about enzymes from this organism that represent potential drug targets. The gene for P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) has been cloned and the protein expressed as a hexahistidine-tagged recombinant protein in Escherichia coli. The recombinant protein has been crystallized and its three-dimensional structure determined. One molecule of the cofactor NAD+ is bound to each of the four subunits in the tetrameric enzyme. The major structural feature distinguishing human GAPDH from PfGAPDH is the insertion of a dipeptide (-KG-) in the so-called S loop. This insert, together with other characteristic single-amino-acid substitutions, alters the chemical environment of the groove that encompasses the R dyad and that links adjacent cofactor-binding sites and may be responsible for the selective inhibition of the enzyme by ferriprotoporphyrin IX.

Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification

A surface plasmon resonance imaging-based Ni(2+)-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.

Intracellular autoregulation of the Mycobacterium tuberculosis PrrA response regulator.

Two-component systems are major regulatory systems for bacterial adaptation to environmental changes. During the infectious cycle of Mycobacterium tuberculosis, adaptation to an intracellular environment is critical for multiplication and survival of the micro-organism within the host. The M. tuberculosis prrA gene, encoding the regulator of the two-component system PrrA-PrrB, has been shown to be induced upon macrophage phagocytosis and to be transiently required for the early stages of macrophage infection. In order to study the mechanisms of regulation of the PrrA-PrrB two-component system, PrrA and the cytoplasmic part of the PrrB histidine kinase were produced and purified as hexahistidine-tagged recombinant proteins. Electrophoretic mobility shift assays indicated that PrrA specifically binds to the promoter of its own operon, with increased affinity upon phosphorylation. Moreover, induction of fluorescence was observed after phagocytosis of a wild-type M. tuberculosis strain containing the gfp reporter gene under the control of the prrA-prrB promoter, while this induction was not seen in a prrA/B mutant strain containing the same construct. These results indicate that the early intracellular induction of prrA depends on the autoregulation of this two-component system.

Functional polymer affinity matrix for purifying hexahistidine-tagged recombinant protein

A functional polyacrylic acid (PAA) adsorbent has been prepared for metal chelate affinity chromatography. It has been found to chelate nickel ion Ni 2+ strongly, and was evaluated for the ability to bind proteins containing neighbouring histidine residues. The principle of the technique was illustrated with Aeromonas hydrophila outer membrane protein OmpTS. DNA elements coding for adjacent histidines were fused to the Aeromonas hydrophila ompTS gene. Subsequent expression in E. coli resulted in the production of hybrid protein His 6-OmpTS that could be purified by Ni 2+–PAA affinity chromatography. The remarkable specificity found makes it an attractive addition to the range of adsorbents for metal chelate affinity chromatography.

Detection and Quantitation of Hexa-Histidine-Tagged Recombinant Proteins on Western Blots and by a Surface Plasmon Resonance Biosensor Technique

The use of short peptide affinity tag sequences has become commonplace for the expression and purification of recombinant proteins. Many of these tags are antibody epitopes and detection of tagged proteins via Western blots is straightforward. However, the most common affinity tag used at present for the expression of recombinant proteins is a hexa-histidine, or like sequence, which exhibits strong affinity for Ni(II). The one drawback of histidine-containing affinity tags is the inability to specifically detect such recombinant proteins on Western blots. Here we describe the synthesis and use of biotinyl-nitrilotriacetic acid which, in combination with streptavidin-horseradish peroxidase, allows for the detection of hexa-histidine-tagged recombinant proteins on Western blots. In addition, we describe a surface plasmon resonance technique, employing a solid-phase Ni(II)-nitrilotriacetic acid complex, for the detection and quantitation of hexahistidine-tagged recombinant proteins in solution. The surface plasmon resonance technique also allows for the oriented immobilization of the recombinant proteins for subsequent ligand interaction studies.