IMAC capture of recombinant protein from unclarified mammalian cell feed streams

Fusion‐tag affinity chromatography is a key technique in recombinant protein purification. Current methods for protein recovery from mammalian cells are hampered by the need for feed stream clarification. We have developed a method for direct capture using immobilized metal affinity chromatography (IMAC) of hexahistidine (His6) tagged proteins from unclarified mammalian cell feed streams. The process employs radial flow chromatography with 300–500 μm diameter agarose resin beads that allow free passage of cells but capture His‐tagged proteins from the feed stream; circumventing expensive and cumbersome centrifugation and/or filtration steps. The method is exemplified by Chinese Hamster Ovary (CHO) cell expression and subsequent recovery of recombinant His‐tagged carcinoembryonic antigen (CEA); a heavily glycosylated and clinically relevant protein. Despite operating at a high NaCl concentration necessary for IMAC binding, cells remained over 96% viable after passage through the column with host cell proteases and DNA detected at ～8 U/mL and 2 ng/μL in column flow‐through, respectively. Recovery of His‐tagged CEA from unclarified feed yielded 71% product recovery. This work provides a basis for direct primary capture of fully glycosylated recombinant proteins from unclarified mammalian cell feed streams. Biotechnol. Bioeng. 2016;113: 130–140. © 2015 Wiley Periodicals, Inc.     

固定化金属螯合亲和膜纯化重组抗菌肽研究

用自行制备的固定化金属螯合亲和膜对N端含六个组氨酸标记的重组抗菌肽进行了分离纯化,并较好地解决了金属离子泄露问题.实验表明,固定化金属螯合亲和膜性能优于传统琼脂糖凝胶介质,完全适用于分离纯化含有多组氨酸标记的重组蛋白质.     

Expression of recombinant chinese bovine enterokinase catalytic subunit in P. pastoris and its purification and characterization

EK (enterokinase) is a serine proteinase which consistsof a heavy chain and a light chain linked by a disulfidebond. The light chain of EK contains a chymotrypsin-likeserine proteinase domain sufficient for the normal recog-nition and cleavage of EK subst…     

Histidine-tag-directed chromophores for tracer analyses in the analytical ultracentrifuge

Many recombinant proteins carry an oligohistidine (His(X))-tag that allows their purification by immobilized metal affinity chromatography (IMAC). This tag can be exploited for the site-specific attachment of chromophores and fluorophores, using the same metal ion-nitrilotriacetic acid (NTA) coordination chemistry that forms the basis of popular versions of IMAC. Labeling proteins in this way can allow their detection at wavelengths outside of the absorption envelopes of un-modified proteins and nucleic acids. Here we describe use of this technology in tracer sedimentation experiments that can be performed in a standard analytical ultracentrifuge equipped with absorbance or fluorescence optics. Examples include sedimentation velocity in the presence of low molecular weight chromophoric solutes, sedimentation equilibrium in the presence of high concentrations of background protein and selective labeling to simplify the assignment of species in a complex interacting mixture.     

Isolation of carcinoembryonic antigen N-terminal domains (N-A1) from soluble aggregates

Carcinoembryonic antigen (CEA) was identified as a prominent tumor-associated antigen in human colorectal cancer and it is still intensively investigated. However, its physiological role remains unclear. The CEA molecule is composed of seven highly hydrophobic, immunoglobulin-like domains, six of which contain a single disulphide bridge. The production of recombinant protein containing Ig-like domains in bacterial expression systems often results in partial degradation or insolubility due to aggregation hampering the analysis of their native structure and function. Here, we present a new method of expression and purification of CEA N-terminal domains (N-A1) fused to MBP in Escherichia coli. In order to optimize the expression and purification of CEA N-A1 domains we evaluated bacteria cultivation conditions, the length of N-A1 domains, fusion systems (GST- and MBP-tag), IPTG concentrations and protein purification conditions. We have found that MBP-N-A1 fusion protein digested with TEV protease forms soluble aggregates composed of N-A1 domains and incompletely digested MBP-N-A1 fusion protein. Using 1.25 M guanidinium chloride (GdmCl) as a component of the elution buffer we were able to achieve an almost complete dissociation of the aggregates. The dissociation was monitored by circular dichroism and fluorescence measurements. The CD spectra and Ellman's assay suggest that the conformation of N-A1 domains and their disulphide bonds are correct.     

Expression and purification of neurolin immunoglobulin domain 2 from Carrassius auratus (goldfish) in Escherichia coli

The immunoglobulin superfamily protein neurolin plays a central role during differentiation and development of retina ganglion cells in goldfish. As shown in earlier work, blockage of the second immunoglobulin domain (Ig2) of neurolin with domain-specific antibodies causes severe pathfinding defects of growing axons in the retina. Thus Ig2 of neurolin was identified as the critical domain for axon guidance. In the present study we have developed a protocol for expression and purification of neurolin-Ig2 suitable for structure analysis, functional studies and ligand identification. Neurolin was expressed in Rosettagami and Origami strains of Escherichia coli which is deficient in glutathione- and thioredoxin reductase facilitating proper formation of the disulfide bond in the cytoplasm. The protein was purified via an N-terminal His(6)-tag by Ni(2+) affinity and size exclusion chromatography. After purification the His(6)-tag was cut-off without loss of solubility. Analytical size exclusion chromatography revealed an apparent molecular mass for neurolin-Ig2 in agreement with a non-covalent homodimer. Analysis of CD and FTIR spectra gave a secondary structure content typical for Ig domains.     

Polymer versus monomer as displacer in immobilized metal affinity chromatography

Successful immobilized metal affinity chromatography (IMAC) of proteins on Cu²⁺-iminodiacetic acid Sepharose has been carried out in a displacement mode using a synthetic copolymer of vinyl imidazole and vinyl caprolactam [poly(VI-VCL)] as a displacer. Vinyl caprolactam renders the co-polymer with the thermosensitivity, e.g., property of the co-polymer to precipitate nearly quantitatively from aqueous solution on increase of the temperature to 48°C. A thermostable lactate dehydrogenase from the thermophilic bacterium Bacillus stearothermophilus modified with a (His)₆-tag [(His)₆-LDH] has been purified using an IMAC column. For the first time it was clearly demonstrated that a polymeric displacer [poly(VI-VCL)] was more efficient compared to a monomeric displacer (imidazole) of the same chemical nature, probably due to the multipoint interaction of imidazole groups within the same macromolecule with one Cu²⁺ ion. Complete elution of bound (His)₆-LDH has been achieved at 3.7 mM concentration of imidazole units of the co-polymer (5 mg/ml), while this concentration of free imidazole was sufficient to elute only weakly bound proteins. Complete elution of (His)₆-LDH by the free imidazole was achieved only at concentrations as high as 160 mM. Thus, it was clearly demonstrated, that the efficiency of low-molecular-mass displacer could be improved significantly by converting it into a polymeric displacer having interacting groups of the same chemical nature.     

Membrane-bound human 3beta-hydroxysteroid dehydrogenase: overexpression with His-tag using a baculovirus system and single-step purification

The membrane-bound human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) was overexpressed with His(6)-tag, using a baculovirus expression system, and then purified by nickel-chelated affinity chromatography. Overexpression of 3beta-HSD1 was confirmed by enzyme assay and Western blot analysis. The protein was purified to more than 95% homogeneity by a single-step Ni(2+)-chelated affinity chromatography after solubilization of the membrane-bound protein with the detergent C(12)E(8). High yield was repeatedly obtained, with 3-4 mg of homogeneous and active 3beta-HSD1 from 1 x 10(9) of infected Sf9 cells. The kinetic study showed a K(m) of 1.7 microM and a V(max) of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substrate. The above preparation will facilitate the structure-function study of this important enzyme.     

Overexpression of juvenile hormone binding protein in bacteria and Pichia pastoris

Galleria mellonella juvenile hormone binding protein (JHBP) is a single chain glycoprotein with two disulfide bonds and a molecular mass of 25,880 Da. This report describes the expression of JHBP in bacteria and yeast cells (Pichia pastoris). The expression in bacteria was low and the protein was rapidly degraded upon cell lysis. The expression of His8-tagged rJHBP (His8-rJHBP) in P. pastoris was high and the non-degraded protein was purified to homogeneity with high yield in a one-step immobilized Ni++ affinity chromatography. His8-rJHBP from P. pastoris contains one JH III binding site with KD of 3.7 +/- 1.3x10(-7) M. The results suggest that P. pastoris is the preferred system for expression of His8-rJHBP in non-degraded fully active form.     

Purification of the human apical conjugate export pump MRP2 reconstitution and functional characterization as substrate-stimulated ATPase.

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.     

Expression, purification, and characterization of rat protein tyrosine phosphatase eta catalytic domain

Receptor-like protein tyrosine phosphatases generally contain one or two conserved intracellular catalytic domains with a conserved sequence motif ([I/V]HCXAGXXR[S/T]G), a single transmembrane domain, and an external highly variable part. Here, we describe cloning of the intracellular catalytic domain of the rat protein tyrosine phosphatase eta (rPTPetaCD) into pET28a(+) vector, its expression in Escherichia coli, purification and initial characterization. The purification of His6-tagged rPTPetaCD to near homogeneity was achieved by a combination of affinity and size exclusion chromatography. The His-tag was subsequently removed by thrombin digestion. PhastGel IEF electrophoresis demonstrated that the isoelectric point of this 41 kDa His6-tag free recombinant protein was 7.3, which is just slightly higher than the theoretically predicted value of 7.2. To assess the functionality of the rPTPetaCD we used the pNPP hydrolysis assay and observed that the enzyme has a specific activity of 9 nmol/min/mug. The secondary structure and stability of the recombinant protein was also analyzed by circular dichroism and fluorescence spectroscopy. In summary, the rPTPetaCD is stable at 18 degrees C, properly folded, and fully active, which makes it a suitable candidate for structural and functional studies.     

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct

Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20-25% binding whereas both His6-scFv and scFv (no His-tag) showed 60-65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (KA) for His6-scFv and scFv as 1.19 x 10(6) M(-1) and 3.27 x 10(6) M(-1), respectively. No K(A) value could be calculated for scFv-His6 due to poor binding kinetics (kon and koff). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.     

Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.     

Highly-efficient purification of native polyhistidine-tagged proteins by multivalent NTA-modified magnetic nanoparticles

A new bis-nitrilotriacetic acid (NTA) chelate with catechol anchor was synthesized and immobilized on superparamagnetic iron oxide nanoparticles. When loaded with Ni(II), these bis-NTA-immobilized nanoparticles were shown to bind polyhistidine (His x 6-tagged) fusion proteins in their native, folded conformations that commercial microbeads failed to bind under identical conditions. Control experiments with a mono-NTA chelate immobilized on iron oxide nanoparticles indicate a similarly high affinity for His x 6-tagged native proteins, suggesting that the high density of the mono-NTA chelate presented by the nanoparticles allows the binding of the His x 6-tag to more than one Ni-NTA moiety on the surface. This study shows that the multivalency strategy can be utilized to enhance the binding of His x 6-tagged proteins in their native, folded conformations. We further demonstrated the selective purification of His x 6-tagged proteins from crude cell lysates by using the Ni(II)-loaded iron oxide nanoparticles. The present platform is capable of efficient purification of His x 6-tagged proteins that are expressed at low levels in mammalian cells. This work thus presents a novel nanoparticle-based high-capacity protein purification system with shorter incubation times, proportionally large washes, and significantly smaller elution volumes compared to commercially available microbeads.     

High-level expression and single-step purification of leucyl-tRNA synthetase from Escherichia coli

A T7 promoter-based His6-tagging vector has been constructed that directs the synthesis in Escherichia coli of fusion proteins containing a stretch of six histidine residues at the N terminus. The vector allows overproduction and single-step purification of His6-fusion protein by immobilized metal (Ni2+) chelate affinity chromatography. The gene encoding leucyl-tRNA synthetase (leuS) was cloned into this vector and expressed in high level. The specific activity of the synthetase in the crude extract of E. coli JM109(DE3) transformant containing the His6-tagging vector with the gene leuS was approximately 110 times that of JM109(DE3) (the host strain without the vector). The overproduced His6-fusion leucyl-tRNA synthetase can be purified to homogeneity under native conditions within 2 h by one-step affinity chromatography with an overall yield of 55%. The His6-tag at the N terminus of leucyl-tRNA synthetase did not affect its aminoacylation activity or the secondary structure.     

Production and characterization of clinical grade Escherichia coli derived Plasmodium falciparum 42 kDa merozoite surface protein 1 (MSP1(42)) in the absence of an affinity tag

The 42 kDa cleavage product from the carboxyl end of the Plasmodium falciparum merozoite surface protein 1 (MSP1(42)) is an important blood-stage malaria vaccine target. Several recombinant protein expression systems have been used for production of MSP1(42) including yeast (Saccharomyces cerevisiae and Pichia pastoris), Escherichia coli, baculovirus and transgenic animals. To date, all of the reported recombinant proteins include a 6 x His affinity tag to facilitate purification, including three MSP1(42) clinical grade proteins currently in human trials. Under some circumstances, the presence of the 6 x His tag may not be desirable. Therefore, we were interested to produce clinical grade MSP1(42) without a 6 x His affinity tag from E. coli inclusion bodies. We produced a recombinant MSP1(42) with a P. falciparum FUP (Uganda-Palo Alto) phenotype which accounts for a substantial proportion of the MSP1(42) protein observed in African isolates. EcMSP1(42)-FUP was produced in E. coli inclusion bodies by high cell mass induction with IPTG using 5 L and 60 L bioreactors. Isolated inclusion bodies were solubilized in 8M guanidine-HCl and the EcMSP1(42)-FUP protein refolded by rapid dilution. Refolded EcMSP1(42)-FUP was purified using hydrophobic interaction chromatography, anion exchange chromatography, and size exclusion chromatography, and subject to biochemical characterization for integrity, identity, and purity. Endotoxin and host cell protein levels were within acceptable limits for human use. The process was successfully transferred to pilot-scale production in a cGMP environment. A final recovery of 87.8 mg of clinical-grade material per liter of fermentation broth was achieved. The EcMSP1(42)-FUP clinical antigen is available for preclinical evaluation and human studies.     

High-throughput Purification and Quality Assurance of Arabidopsis thaliana Proteins for Eukaryotic Structural Genomics

The Center for Eukaryotic Structural Genomics (CESG) has established procedures for the purification of Arabidopsis proteins in a high-throughput mode. Recombinant proteins were fused with (His)(6)-MBP tags at their N-terminus and expressed in Escherichia coli. Using an automated AKTApurifier system, fusion proteins were initially purified by immobilized metal affinity chromatography (IMAC). After cleavage of (His)(6)-MBP tags by TEV protease, (His)(6)-MBP tags were separated from target proteins by a subtractive 2nd IMAC. As a part of quality assurance, all purified proteins were subjected to MALDI-TOF and ESI mass spectrometry to confirm target identity and integrity, and determine incorporation of seleno-methionine (SeMet) and (15)N and (13)C isotopes. The protocols have been used successfully to provide high quality proteins that are suitable for structural studies by X-ray crystallography and NMR.     

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.     

Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli

Barley alpha-amylase/subtilisin inhibitor (BASI) is a beta-trefoil fold protein related to soybean trypsin inhibitor (Kunitz) and inhibits barley alpha-amylase isozyme 2 (AMY2), which is de novo synthesized in the seed during germination. Recombinant BASI was produced in Escherichia coli in an untagged form (untagged rBASI), in two His(6)-tag forms (His(6)-rBASI and His(6)-Xa-rBASI), and in an intein-CBD-tagged form (rBASI (intein)). The yields per liter culture after purification were (i) 25 mgl(-1) His(6)-rBASI; (ii) 6 mgl(-1) rBASI purified after cleavage of His(6)-Xa-rBASI by Factor Xa; (iii) 3 mgl(-1) untagged rBASI; and (iv) 0.2 mgl(-1) rBASI after a chitin-column and autohydrolysis of the rBASI-intein-CBD. In Pichia pastoris, rBASI was secreted at 0.1 mgl(-1). The recombinant BASI forms and natural seed BASI (sBASI) all had an identical isoelectric point of 7.2 and a mass of 19,879 Da, as determined by mass spectrometry. The fold of rBASI from the different preparations was confirmed by circular dichroism spectroscopy and rBASI (intein), His(6)-rBASI, and sBASI inhibited AMY2 catalyzed starch hydrolysis with K(i) of 0.10, 0.06, and 0.09 nM, respectively. Surface plasmon resonance analysis of the formation of AMY2/rBASI (intein) gave k(on)=1.3x10(5)M(-1)s(-1), k(off)=1.4x10(-4)s(-1), and K(D)=1.1 nM, and of the savinase-His(6)-rBASI complex k(on)=21.0x10(4)M(-1)s(-1), k(off)=53.0x10(-4)s(-1), and K(D)=25.0 nM, in agreement with sBASI values. K(i) was 77 and 65 nM for inhibition of savinase activity by His(6)-rBASI and sBASI, respectively.     

友菌素核苷转移酶amiE基因的克隆、表达和功能鉴定

【目的】克隆和表达二糖核苷类抗生素友菌素生物合成基因簇中的核苷转移酶基因amiE,并研究AmiE的体外催化功能。【方法】采用PCR技术将编码257个氨基酸的葡萄糖-1-磷酸核苷转移酶基因amiE克隆到表达载体pET28a上,构建质粒pCSG4001,转化入大肠杆菌E.coli BL21(DE3)中诱导表达;利用亲和层析分离纯化蛋白AmiE,以葡萄糖-1-磷酸和胸腺嘧啶三磷酸(TTP)或尿嘧啶三磷酸(UTP)为底物,利用高效液相检测AmiE的体外酶活;以甘露糖-1-磷酸、半乳糖胺-1-磷酸和半乳糖-1-磷酸和TTP作为底物,进一步研究AmiE对底物的选择性。【结果】N-末端融合组氨酸标签的AmiE蛋白在大肠杆菌中获得了可溶性表达,通过亲和层析纯化出的AmiE能够以TTP(或UTP)和葡萄糖-1-磷酸作为底物,催化形成胸腺嘧啶二磷酸葡萄糖(TDP-glucose)或者尿嘧啶二磷酸葡萄糖(UDP-glucose),但对其他三种底物,无明显催化活性。【结论】大肠杆菌中表达纯化的核苷转移酶AmiE能够体外催化形成TDP-葡萄糖(或UDP-葡萄糖),确证了AmiE作为核苷转移酶的催化功能,同时表明AmiE对底物具有一定的选择性。