Journal of Chromatography B (v.818, #1)

Preface by Xavier Santarelli (1).

Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS–PAGE.
Keywords: CELBEADS; DNA; Hydrophobic interaction chromatography; Plasmid;

Preparative parallel protein purification (P4) by Patrik Strömberg; Joke Rotticci-Mulder; Robert Björnestedt; Stefan R. Schmidt (11-18).
In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an ÄKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.
Keywords: Automation; Protein purification; Affinity chromatography;

LdARL-1 His-tagged recombinant protein: purification by immobilized metal affinity expanded bed adsorption by Annelise Sahin; Emmanuel Tetaud; Gilles Merlin; Xavier Santarelli (19-22).
Previously we have cloned three ADP-ribosylation factor-like (ARL) genes from the parasitic protozoan Leishmania donovani: LdARL-3A and 3B, LdARL-1. LdARL-3A was previously purified as an active native form, which was able to bind GTP in vitro. In this paper, we have performed the production and the purification of Histidine-tagged (His-tagged) LdARL-1 recombinant protein by immobilized metal affinity chromatography (IMAC) using expanded bed adsorption (EBA) technology. This protein was purified with more than 95% purity and could be successfully used for GTP-binding assay.
Keywords: LdARL-1; ADP-ribosylation factor-like protein; His-tagged recombinant protein; Immobilized metal affinity chromatography (IMAC); Expanded bed adsorption;

The enhanced green fluorescent protein (EGFP) was over-expressed in Escherichia coli as inclusion bodies to increase its quantity and to facilitate its purification. Insoluble EGFP has been purified on Q Hyper Z matrix by expanded bed adsorption after solubilization in 8 M urea. The adsorption was made in expanded bed mode to avoid centrifugation. EBA-column refolding was done by elimination of urea and elution with NaCl. The EGFP was obtained as a highly purified soluble form with similar behavior in fluorescence and electrophoresis as native EGFP.
Keywords: Expanded bed adsorption; EGFP; Purification; Chromatography; Refolding; Inclusion bodies;

Expanded bed absorption chromatography (EBA) was used to improve and simplify the purification of several wheat recombinant proteins. Binding and elution conditions were set to allow the purification of the over expressed protein in a single step. In comparison with our previous multi step protocol, same purity was obtained while EBA required less time (one day instead of five) and gave a higher yield (63% instead of 10%). This new procedure was then used for the successful purification of five other wheat ns-LTP. Despite their important polymorphism (identity from 44 to 97 %—pHi from 8 to 10), the EBA protocol allowed their purification in a single step.
Keywords: Recombinant protein purification; Expanded bed absorption chromatography; Pichia pastoris; Wheat ns-LTP;

Large scale purification of rapeseed proteins (Brassica napus L.) by S. Bérot; J.P. Compoint; C. Larré; C. Malabat; J. Guéguen (35-42).
Rapeseed (Brassica napus L.) cruciferin (12S globulin), napin (2S albumin) and lipid transfer proteins (LTP) were purified at a multi-g scale. The procedure developed was simple, rather fast and resolutive; it permitted the recovery of these proteins with a good yield, such as 40% for cruciferin and 18% for napin. Nanofiltration eliminated the major phenolic compounds. The remaining protein fraction was fractionated by cation exchange chromatography (CEC) on a streamline SP-XL column in alkaline conditions. The unbound neutral cruciferin was polished by size exclusion chromatography. The alkaline napin isoforms and LTP, adsorbed on the beads, were eluted as a whole fraction and further separated by an other CEC step at acidic pH. Napins were polished by hydrophobic interaction chromatography (HIC). The fractions were characterized by reverse phase HPLC, electrophoresis, N-terminal sequencing and mass spectrometry. All the fractions contained less than 5% of impurities.
Keywords: Purification; Preparative scale; Rapeseed; Cruciferin; Napin; LTP;

Improvement in production and purification bioprocesses of bacterially expressed anti-alphaIIbbeta3 human single-chain FV antibodies by R. Robert; A.M. Noubhani; Marie-Josée Jacobin; X. Santarelli; G. Clofent-Sanchez (43-51).
Production of anti-alphaIIbbeta3 (anti-αIIbβ3)-binding single-chain FV (scFv) fragments obtained from combinatorial libraries of IgG human antibodies is of broad interest for imaging and treatment of acute coronary syndromes. The objective of our work was to design an optimized production of one selected anti-αIIbβ3-binding scFv fragment for subsequent in vivo animal studies. Fed-batch fermentation was initiated with 2TY media supplemented with 0.1 M glucose. This growing batch culture was used as a starting point for further fed-batch induction, in which a media without glucose containing 1 mM IPTG and 0.4 M saccharose was continuously added. Subsequent purification was performed on the whole cell extract in native conditions over an immobilized copper-ion affinity column. The improved conditions allowed the recovery of 5 mg of highly purified scFv fragments as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the scFv fragments was further monitored by ELISA, cytometric and immunohistochemical methods.
Keywords: IMAC purification; Single-chain Fv antibody fragment; AlphaIIbbeta3 integrin; Fermentation;

We developed the synthesis of new supports for the purification of insulin and IgG by affinity chromatography. The preparation of such an affinity support is performed in two steps. First, silica beads are coated with dextran polymers carrying a calculated amount of positively charged diethylaminoethyl groups in order to mask negative charges at its surface. Second, ligand is immobilized using a coupling agent. This support combines the advantages of polysaccharide phases with the excellent mechanical characteristics of silica. The existence of N-acetylneuraminic acid (sialic acid) in insulin receptor and in the antigenic determinant of IgG suggests that such an acid may develop specific interactions usable in affinity chromatography. Therefore, N-acetylneuraminic acid was used as an active ligand. The immobilization of sialic acid can be carried out by using the conventional coupling agent: the carbonyldiimidazole. The performances of these supports grafted by sialic acid were studied by high-performance liquid affinity chromatography (HPAC). The optimization of the chromatographic conditions (support characteristics and mobile phase) enabled us to observe a behavior of the type “thiophilic” of the support, which does not contain sulfone group. This new affinity support allowed a one-step separation of the IgG from mouse ascitic fluids and also allowed the insulin purification from a pancreatic extract with a good purification yields.
Keywords: Silica; HPAC; Thiophilic interactions; N-Acetylneuraminic acid; Insulin; IgG;

A synthetic ligand called 2-mercapto-5-benzimidazolesulfonic acid has been successfully used for the specific chromatographic capture of antibodies from a cell culture supernatant. Adsorption occurred at physiological ionic strength and pH range between 5.0 and 6.0, with some binding capacity variations within this pH range: antibody uptake increased when the pH decreased. With very dilute feedstocks, as was the case with the cell culture supernatant under investigation, it was found that the pH had to be slightly lowered to get a good antibody sorption capacity. To optimize separation conditions, a preliminary study was made using ProteinChip® Arrays that displayed the same chemical functionalities as the resin. Arrays were analyzed using SELDI–MS. By this mean, it was possible to cross-over simultaneously different pH conditions at the adsorption and the desorption steps. Best conditions were implemented for preparative separation using regular lab-scale columns. At pH 5.2, antibody adsorption was not complete, while at pH 5.0 the antibody was entirely captured. pH 9 was selected at elution, rather than pH 8.0 or 10.0, and resulted in a complete desorption of antibodies from the column. Benefits of the prediction of separation conditions of antibodies on MBI beads using SELDI–MS were a significant reduction in analysis time and in sample volume. This was possible because the separation of IgG on the chip surface did mimic very well the separation on beads.
Keywords: Antibodies; 2-Mercapto-5-benzimidazolesulfonic acid; Affinity chromatography; ProteinChips®; Surface enhanced laser desorption ionisation–time of flight–mass spectrometry (SELDI–TOF–MS);

A peptide of around 7.4 kDa has been purified from the aqueous extract of human placenta used as wound healer. Derived partial amino acid sequence from mass spectrometric analysis showed its homology with human fibronectin type III. Under nondenaturing condition, it formed aggregate, the elution pattern of which from reverse-phase HPLC was identical with that of fibronectin type III. Immuno-blot of the peptide with reference fibronectin type III-C showed strong cross reactivity. Since fibronectin type III plays important roles in wound healing, similar peptide in the extract is likely to take part in curing process.
Keywords: Human placental extract; Fibronectin type III; HPLC; Amino acid sequence; Dot blot;

Copper binding to prion octarepeat peptides, a combined metal chelate affinity and immunochemical approaches by Daniela Todorova-Balvay; Stéphanie Simon; Christophe Créminon; Jacques Grassi; Thamarapu Srikrishnan; Mookambeswaran A. Vijayalakshmi (75-82).
Based on the hypothetical proposal of Sulkowski [E. Sulkowski, FEBS Lett. 307 (2) (1992) 129] for the implication of transition metal ions in the structural changes/oligomerisation of normal cellular prion protein (PrPc) resulting in the pathological isoform (PrPsc), we focused our study on the octarepat domain of this protein which has been supposed to be the metal binding site. We have studied the copper binding to synthetic prion octarepeat peptides (PHGGGWGQ)n (n  = 1, 3, 6) using metal chelate and size-exclusion modes of chromatographies. This copper binding induces oligomerisation resulting in multiple aggregates. Moreover, heterogeneity of metal bound octarepeat oligomers by ESI-MS has been demonstrated. In addition, anti prion antibodies specific to the octarepeat region were used to discriminate between metal free and copper, nickel and zinc bound hexamer octarepeat peptide. Differential recognition of Cu(II) and Zn(II) bound complexes has been observed which signify differences in exposed epitopes of aggregated peptides.
Keywords: Prion; Octarepeat peptides; Copper; Metal chelate affinity; Mass spectrometry; Anti-prion peptide antibody;

One step flow-through adsorptive purification of tubulin from tissue homogenate by Annamma Anil; Reena Pandit; Madhavi Indap; Arvind Lali (83-87).
Tubulin, a potential target for anti-cancer drugs, has been purified in one step and obtained as flow-through fraction directly from an extract of a mammalian brain tissue by adsorption chromatography on H-CELBEADS, an indigenously developed rigid, superporous cross-linked cellulose based weakly hydrophobic adsorbent. The fibrous polymerized tubulin mass passed through the H-CELBEADS bed while the associated proteins were separated by adsorption. The final tubulin preparation was obtained free from other proteins as seen on SDS–PAGE. Purified tubulin was obtained in a yield of about 29 mg/100 g brain, and its bioactivity, evaluated through its ability to bind colchicine, was found to be preserved.
Keywords: CELBEADS; Protein purification; Tubulin;

Large-pore materials or supports resembling polymer conduits are used as packing material in chromatographic operations. Our ongoing research has shown that, when modified with peptides or ligands, chitosan beads that are 800 μm in diameter and have 3.5% solids can be used as matrices in bioseparations. The goal of the present study is to evaluate the transport properties of biomolecules in the modified chitosan beaded matrices. Batch uptake experiments with fluorescently tagged pure human IgG, human IgA and human IgM were conducted to visualize the distribution of binding sites throughout the bead as well as to evaluate restrictions to diffusion, if any, within the support. The chromatographic performance of the macrobeads was first assessed by the classical height equivalent of a theoretical plate HETP analysis. The independence of HETP on linear flow rates studied suggests that a likely mode of solute transport within the macrobeads may be a combination of convection and diffusion-convective components. By using fluorescent-tagged immunoglobulins, the penetration of the adsorbent particle at different times and different levels of saturation was visually observed. The profiles obtained from dynamic experiments were compared to the profiles obtained from finite bath experiments. With an increase in the incubation time, the degree of penetration increased and the bead interior was saturated with FITC immunoglobulins at the end point of the finite bath experiment. In the dynamic uptake experiment, the degree of penetration was found to be a function of the linear velocity and level of breakthrough. The penetration of the bead radius, at times lower than the predicted diffusion time, suggests that the mode of transport in the chitosan beads is governed by a combination of convective and diffusive forces.
Keywords: Protein transport; Biomolecules; Convection; Confocal microscopy; Diffusion; FITC;

Carrier ampholytes as potential buffers in electrophoresis: physico-chemical study by Jean-Marc Busnel; Marie-Claire Hennion; Gabriel Peltre (99-107).
Joule heating is a limiting factor when separating proteins in capillary zone electrophoresis (CZE). Low conductivity buffers, are required for high-speed separations. We investigated the use of carrier ampholytes (CA) as background electrolytes (BGE) in CZE. We prepared 25 “narrow pH cuts” of wide pH range (3–10) CA mixture in order to know if these fractions were suitable to be used as BGE in CZE. Each fraction was characterised by CZE analysis, giving an idea of its heterogeneity (number and relative abundance of molecular ampholytes). Conductivities and buffering capacities of each fraction have been also measured. Our conclusion is that “narrow pH cuts” of CA might be well suited buffers for electrophoretic separations.
Keywords: Carrier ampholytes; Background electrolyte; Conductivity; Buffering capacity;