Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand

The fusion protein of green fluorescent protein (GFP) and human interleukin-2 (hIL-2) was produced in insect Trichoplusia ni larvae infected with recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus (AcNPV). This fusion protein was composed of a metal ion binding site (His)6 for rapid one-step purification using immobilized metal affinity chromatography (IMAC), UV-optimized GFP (GFPuv), enterokinase cleavage site for recovering hIL-2 from purified fusion protein, and hIL-2 protein. The additional histidine residues on fusion protein enabled the efficient purification of fusion protein based on immobilized metal affinity chromatography. In addition to advantages of GFP as a fusion marker, GFP was able to be used as a selectable purification marker; we easily determined the correct purified fusion protein sample fraction by simply detecting GFP fluorescence.     

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.     

Purification of insect vitellogenin and vitellin by gel-immobilized ferric chelate.

Vitellogenin and vitellin of Manduca sexta and some other insect species were purified by immobilized metal ion affinity chromatography. Ferric ion was chosen as the immobilized metal ion. Agarose-bound carboxymethylpicolylamine was used as the chelating adsorbent for the ferric ion. Vitellogenin and vitellin, both phosphorylated lipoproteins, were shown to bind specifically to the iron. The general applicability of immobilized ferric ion affinity chromatography for the purification of insect vitellogenin and vitellin is suggested.     

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.     

A method for large scale purification of turnip peroxidase and its characterization

Purification of peroxidase has been carried out since 1960 from different sources and with different methods. Ion exchange, affinity, hydrophobic, and metal affinity chromatography are known, to our knowledge. The present method, developed in this study, is three-phase partitioning, a novel technique to separate protein directly from a large volume of crude suspension. It has been observed that interfacing phase with a metal makes this technique highly selective. Turnip peroxidase purified with this method has 512 units/mg with 20.3% recovery. The natural proteins containing histidine or cystine are often purified by immobilized metal affinity chromatography. The purification of turnip peroxidase with the three-phase partitioning technique is based on immobilized metal affinity chromatography and is used for large-scale purification. The present method, described here, would prove its value in purifying an industrially important enzyme on a large scale from a crude suspension. The enzyme purified with this technique showed two bands on SDS- PAGE, which showed a molecular weight of approx. 39KD. Enzyme showed maximum purification with Cu++ metal and had a maximum activity at pH 6.0. The enzyme has an affinity towards hydrogen peroxide as its substrate in the presence of orthodianisidine as a chromogenic substrate. Enzyme activity was enhanced with calcium and magnesium, whereas sodium, potassium, and manganese inhibit the enzyme activity.     

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.     

Purification of carboxypeptidase B by zinc chelate chromatography

A method is described to purify pancreatic carboxypeptidases B (CPB), removing contaminating endoproteinases that interfere with use of CPB for carboxy-terminal analysis or modification of proteins. The separation uses zinc chelate chromatography and is based on the property that CPB has higher affinity for immobilized zinc ions than do serine proteinases such as trypsin and chymotrypsin, which are abundant endoproteolytic activities in pancreas. CPB preparations are loaded onto a column of iminodiacetic acid-Sepharose that has been saturated with ZnCl2. A step gradient with buffers of decreasing pH is used to elute bound proteins. CPB elutes at a lower pH than do the serine proteinases.     

Purification of human serum amyloid P component (SAP) by calcium affinity chromatography

Serum amyloid P component (SAP) has been purified from human serum by means of immobilized metal ion affinity chromatography (IMAC). It was selectively concentrated on carboxymethylated aspartic acid agarose (CM-Asp-agarose) loaded with calcium and, employing very mild conditions, purified to electrophoretical and immunological homogeneity in a single step amounting to about 1900-fold purification. As a purification method our procedure thus compares well with bio-specific affinity chromatography.     

Bacterial expression of an immunologically reactive PCV2 ORF2 fusion protein

The entire coding region of open reading frame 2 (ORF2) of porcine circovirus 2 (PCV2) was linked to the 3'-end of the maltose-binding protein (MBP)-His(8)-tag gene. The fusion protein was expressed as soluble form after induction by isopropylthio-beta-d-galactoside. MBP-His(8)-ORF2 was purified to homogeneity by immobilized metal affinity chromatography based on the interaction of the polyhistidine-tag with metal ions. Expression could represent 1% of the total protein in Escherichia coli, allowing approximately 1 mg of highly purified protein to be obtained per liter of bacterial culture. The fusion protein was recognized in Western blot by anti-PCV2 polyclonal antibody and swine sera with PCV2 infection. In addition, rabbit polyclonal antibody raised against purified MBP-His(8)-ORF2 fusion protein reacted with the ORF2 protein in immunoprecipitation. The availability of this fusion protein should permit a thorough study of prevalence of PCV2 infection in large-scale serological studies of field samples.     

Purification and Characterization of a Novel Anti-Proliferative Lectin from Morus alba L. Leaves

A novel anti-proliferative lectin was purified from Morus alba L. (Mulberry) leaves by a two step chromatographic procedure namely, immobilized metal ion affinity chromatography (IMAC) and convective interaction media (CIM) based anion exchange chromatography. The purified mulberry leaf lectin (MLL) was specific to galactose, galactosamine and N-acetyl galactosamine (GalNAc). MLL was homogenous with a molecular weight of ~56kDa in silver stained SDS-PAGE. The lectin showed RBC agglutination activity up to 40°C and was independent of pH above pH 6. Haemagglutination activity of purified MLL was not dependent on any metal ions. However, with high concentration of trivalent metal ions, Fe3+ and Al3+ and the divalent metal ion Fe2+, a three fold increase in agglutination activity was observed. The purified MLL showed an anti-proliferative activity towards human breast cancer cells (MCF-7) and colon cancer cells (HCT-15) with a higher potency towards MCF-7 cells. This is the first report on the anti-proliferative activity of a GalNAc specific lectin from M. alba.     

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     

Recombinant expression and purification of functional XorII, a restriction endonuclease from Xanthomonas oryzae pv. oryzae

An endonuclease from Xanthomonas oryzae pathovar oryzae KACC 10331, XorII, was recombinantly produced in Escherichia coli using a T7 system. XorII was purified using a combination of ion exchange and immobilized metal affinity chromatography (IMAC). An optimized washing protocol was carried out on an IMAC in order to obtain a high purity product. The final amount of purified XorII was approximately 2.5 mg/L of LB medium. The purified recombinant XorII was functional and showed the same cleavage pattern as PvuI. The enzyme activity tested the highest at 25 degrees in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, and 1 mM dithiothreitol at a pH of 7.9.     

亲和层析法用于噬菌体抗体库的筛选

本研究报道一种基于固定化金属亲和层析(IMAC)的噬菌体抗体库液相筛选方法。将纯化的带有His标签的抗原与噬菌体抗体库混合,噬菌体抗体与抗原充分结合后再加入亲和介质,使噬菌体抗体抗原复合物通过His标签与介质结合,然后通过充分洗涤去除非特异性噬菌体抗体,最后将特异性噬菌体抗体洗脱下来,感染TG1,进行下一轮筛选。整个筛选过程中抗原与抗体的结合在液相中完成,不仅消除了固相介质对抗原表位的影响,也更有利于噬菌体抗体与抗原的充分作用。将此方法应用于HEV NE2蛋白特异性人源噬菌体抗体的筛选,抗原竞争ELISA,阳性血清阻断,可溶性单链抗体表达检测及测序结果表明,最终获得2个特异性人源抗体。     

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

重组人Fab金属螯合层析法纯化条件的研究

在重组人Fab(rh Fab)表达载体的羧基端插入六个组氨酸, 使其对金属螯合层析介质产生特异性吸附, 可用金属螯合亲和层析法进行分离纯化. 采用自制金属(铜、锌金属离子)螯合层析介质, 以pH和咪唑两种洗脱方法,对rh Fab段的纯化效果进行了探讨. 结果显示: 铜离子螯合层析介质比锌离子螯合层析介质对rh Fab的亲和能力更强; pH洗脱方法的重复性优于咪唑法; 金属铜离子螯合层析法对rh Fab进行一步纯化可得到纯度大于95%的rh Fab产品.     

Immobilized metal affinity chromatography of monoclonal immunoglobulin M against mutant amidase from Pseudomonas aeruginosa

The chromatographic behavior of monoclonal antibodies (MAbs) of immunoglobulin (Ig) M class against mutant (T103I) amidase from Pseudomonas aeruginosa was investigated on immobilized metal chelates. The effect of ligand concentration, the length of spacer arm, and the nature of metal ion were investigated in immobilized metal affinity chromatography (IMAC). The MAbs against mutant amidase adsorbed to Cu(II), Ni(II), Zn(II), Co(II), and Ca(II)-iminodiacetic acid (IDA) agarose columns. The increase in ligand concentration (epichlorohydrin: 30-60 and 1,4-butanediol-diglycidyl ether: 16-36) resulted in higher adsorption to IgM into immobilized metal chelates. The length of spacer arm was found to affect protein adsorption, as longer spacer arm (i.e., 1,4-butanediol-diglycidyl ether) increased protein adsorption of immobilized metal chelates. The adsorption of IgM onto immobilized metal chelates was pH dependent because an increase in the binding of IgM was observed as the pH varied from 6.0 to 8.0. The adsorption of IgM to immobilized metal chelates was the result of coordination of histidine residues to metal chelates that are available in the third constant domain of heavy chain (CH3) of immunoglobulins, as the presence of imidazole (5 mM) in the equilibration buffer abolished the adsorption of IgM to the column. The combination of tailor-made stationary phases for IMAC and a correct design of the adsorption parameters permitted to devise a one-step purification procedure for IgM. Culture supernatants containing IgM against mutant amidase (T103I) were purified either by IMAC on EPI-60-IDA-Co (II) column or by gel filtration chromatography on Sephacryl S-300HR. The specific content of IgM and final recovery of antibody activity exhibited similar values for both purification schemes. The purified preparations of IgM obtained by both schemes were apparently homogeneous on native polyacrylamide gel electrophoresis with a Mr of 851,000 Da. The results presented in this work strongly suggest that one-step purification of IgM by IMAC is a cost-effective and processcompatible alternative to other types of chromatography.     

Purification of proteins containing zinc finger domains using immobilized metal ion affinity chromatography

Heterologous proteins are frequently purified by immobilized metal ion affinity chromatography (IMAC) based on their modification with a hexa-histidine affinity tag (His-tag). The terminal His-tag can, however, alter functional properties of the tagged protein. Numerous strategies for the tag removal have been developed including chemical treatment and insertion of protease target sequences in the protein sequence. Instead of using these approaches, we took an advantage of natural interaction of zinc finger domains with metal ions to purify functionally similar retroviral proteins from two different retroviruses. We found that these proteins exhibited significantly different affinities to the immobilized metal ions, despite that both contain the same type of zinc finger motif (i.e., CCHC). While zinc finger proteins may differ in biochemical properties, the multitude of IMAC platforms should allow relatively simple yet specific method for their isolation in native state.     

Tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography

Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His6 peptide. Display of the His6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His6-tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 microg/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.     

Proteomic analysis of contaminants in recombinant membrane hemeproteins expressed in <Emphasis Type="Italic">E. coli</Emphasis> and isolated by metal affinity chromatography

Contaminating proteins have been identified by “shotgun” proteomic analysis in 14 recombinant preparations of human membrane heme- and flavoproteins expressed in Escherichia coli and purified by immobilized metal ion affinity chromatography. Immobilized metal ion affinity chromatography of ten proteins was performed on Ni²⁺-NTA-sepharose 6B, and the remaining four proteins were purified by ligand affinity chromatography on 2',5'-ADP-sepharose 4B. Proteomic analysis allowed to detect 50 protein impurities from E. coli. The most common contaminant was Elongation factor Tu2. It is characterized by a large dipole moment and a cluster arrangement of acidic amino acid residues that mediate the specific interaction with the sorbent. Peptidyl prolyl-cis-trans isomerase SlyD, glutamine-fructose-6-phosphate aminotransferase, and catalase HPII that contained repeating HxH, QxQ, and RxR fragments capable of specific interaction with the sorbent were identified among the protein contaminants as well. GroL/GroS chaperonins were probably copurified due to the formation of complexes with the target proteins. The Ni²⁺ cations leakage from the sorbent during lead to formation of free carboxyl groups that is the reason of cation exchanger properties of the sorbent. This was the putative reason for the copurification of basic proteins, such as the ribosomal proteins of E. coli and the widely occurring uncharacterized protein YqjD. The results of the analysis revealed variation in the contaminant composition related to the type of protein expressed. This is probably related to the reaction of E. coli cell proteome to the expression of a foreign protein. We concluded that the nature of the protein contaminants in a preparation of a recombinant protein purified by immobilized metal ion affinity chromatography on a certain sorbent could be predicted if information on the host cell proteome were available.