High-performance liquid chromatographic profiling of fluorescent labelled N-glycans on glycoproteins.

Monitoring protein glycosylation is becoming increasingly important as novel recombinant glycoprotein therapeutics, such as glycoprotein hormones, cytokines and clotting factors, are introduced into clinical use. In this report, we describe an HPLC strategy and an improved and simplified pre-column derivatization procedure to profile N-linked glycans obtained from a variety of commercially available glycoproteins as examples. N-Glycans were first released by peptide:N-glycosidase F and labelled with the fluorescent label, 4-aminobenzoic acid by reductive amination. The labelled N-Glycans were then resolved by normal-phase HPLC and the N-glycan profile could be further improved by separating the N-glycans first according to charge by anion-exchange HPLC prior to the normal-phase HPLC. If required, identification of the fractionated derivatized oligosaccharides can be determined by mass spectrometry. The whole profiling process is simple and can be implemented in most laboratories. Because of the high sensitivity, batch glycan-analysis of low-yield recombinant glycoproteins such as samples in ampoules or obtained in the early stage of production development is possible.

Rat interleukin-10: production and characterisation of biologically active protein in a recombinant bacterial expression system.

Interleukin-10 is an anti-inflammatory Th1 immunosuppressive cytokine, the active form of which is a non-covalent homodimer, and which exhibits species-specificity both with respect to structure and biological activity. The rat homologue of IL-10 shares 73% identity with human IL-10 at the amino-acid sequence level, and has, in addition to the two disulphide bonds present in human IL-10, a fifth, unpaired cysteine (cys-149). Preparation of rat IL-10 by bacterial expression followed by solubilisation and refolding in a glutathione redox system, results in a molecule in which cys-149 is almost entirely oxidised, existing either as disulphide dimer or as a mixed disulphide with glutathione, and which has less than 1% of the activity of the native (cys-149-SH) form of the molecule. Site directed mutagenesis of rat IL-10 to replace cys-149 with tyrosine produces a molecule which readily adopts the active conformation upon solubilisation and refolding, and which is recoverable in good yield from bacterial expression systems. Comparison of the biological activities of rat IL-10tyr149 and commercial rat IL-10 preparations confirms that the activity of native-sequence rat IL-10 is either reduced or absent. It is proposed therefore that the biosynthetic analogue rat IL-10tyr149 is a more useful molecule to investigate the biological actions of IL-10 in the rat.

Rat tumour necrosis factor-alpha: expression in recombinant Pichia pastoris, purification, characterization and development of a novel ELISA.

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.

Rat interleukin 6: expression in recombinant Escherichia coli, purification and development of a novel ELISA.

Interleukin 6 (IL-6) is a cytokine involved in many aspects of the acute phase and immune responses. Cloning of rat IL-6 cDNA into the pET-21d expression plasmid under control of a bacteriophage T7 RNA polymerase promoter system allowed isopropylthio-galactopyranoside (IPTG)-inducible production of recombinant rat IL-6 in Escherichia coli. The cloning, expression and purification of rat IL-6 is described. In this expression system, rat IL-6 was produced in insoluble inclusion bodies. The protein was solubilized in 6 M guanidine hydrochloride and refolded in a glutathione redox system. Refolded rat IL-6 was purified to homogeneity using anion-exchange chromatography on SP-Trisacryl. The purified recombinant rat IL-6 had a molecular mass of 21 756.38+/-0.25 Da, which is within 0.01% of the predicted value, taking into account cleavage of the N-terminal methionine residue and the formation of two disulfide bridges. Recombinant rat IL-6 was 2-3-fold more bioactive than the human standard preparation in the B9 hybridoma bioassay. Purified rat IL-6 was used to raise polyclonal antibodies in sheep and these reagents were used to develop a novel rat IL-6 enzyme-linked immunosorbent assay (ELISA). The ELISA is sensitive to 10 pg/ml and has been shown to detect IL-6 in plasma from rats injected with lipopolysaccharide (LPS).

Chemically and conformationally authentic active domain of human tissue inhibitor of metalloproteinases-2 refolded from bacterial inclusion bodies.

The aggregation of recombinant proteins into inclusion bodies is a major problem for expression in bacterial systems. The inclusion bodies must be solubilized and the denatured protein renatured if an active molecule is to be recovered. We have developed such a procedure for the active N-terminal domain of tissue inhibitor of metalloproteinases-2 [TIMP-2-(1-127)], a small mammalian protein containing three disulfide bonds. Conditions for its renaturation were determined by studying the refolding behaviour of reduced and denatured mammalian-cell-expressed TIMP-(1-127) by intrinsic fluorescence. This strategy allows the development of a refolding protocol before generation of a bacterial expression system, and allows rapid and systematic optimization of each refolding variable by assessing its effect on the rate and extent of the refolding reaction. TIMP-(1-127) was expressed at high levels in Escherichia coli, and refolded from TIMP-2-(1-127) inclusion bodies, by means of the method developed with mammalian-cell-expressed protein, to give a refolding efficiency of 30-40% and a final yield of 11-14 mg purified protein/l culture. The chemical structure and conformation of this material was characterized by electrospray mass spectrometry and two-dimensional 1H-NMR; no significant differences were found between it and the native protein. Mass analysis of uniformly 13C-labeled and 15N-labeled protein was used to help identify a mistranslated TIMP-(1-127) contaminant in the purified refolded sample. This technique provides additional information on the nature of the modification and allows a distinction to be made between those modifications that are cell derived, and those that arise from subsequent handling of the protein.