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Prokaryotic production systems have been widely used to manufacture recombinant therapeutic proteins. Economically, the prokaryotic production – especially of small therapeutic molecules – is advantageous compared to eukaryotic production strategies. However, due to the potential endotoxin and host cell protein contamination, the requirements for the purification process are disproportionately higher and therefore more expensive and elaborate to circumvent. For this reason, the goal of this work was to develop and establish a rapid, simple, inexpensive and ‘up-scalable’ production and purification process, using the therapeutic relevant protein anti-EGFR scFv hu225 as model molecule. Configuring high cell density cultivation of Escherichia coli – using the rha-BAD expression system as production platform – a specific product concentration up to 20 mgscFv/gCDW was obtained. By combining freeze-and-thaw, osmotic shock and pH induced host cell protein precipitation, almost 70% of the product was extracted from the biomass. In a novel approach a mixed mode chromatography was implemented as a capturing and desalting step, which allowed the direct application of further ion exchange chromatography steps for purification up to pharmaceutical grade. Thereby, 50% of the produced scFv could be purified within 10 h while maintaining the biological activity.

PEGylation is a common and highly accepted possibility for half-life prolongation of proteins by increasing the hydrodynamic size. The chromatographic purification of PEGylated protein, using PEG (poly-ethylene glycol) of different PEG chain lengths, with the example of lysozyme and a scFv, is described in detail here, and helpful suggestions for the purification of other PEGylated proteins are listed. The relevant characterization methods for PEGylated proteins, important for the successful purification, are also described. The purification starts with a CEX (cation exchange) chromatography leading to about 95 % purity for polishing HIC (hydrophobic interaction chromatography) is described.

In this manuscript we describe the two-step purification of a mono-PEGylated anti-epidermal growth factor receptor (EGFR) single-chain Fv. A weak cation exchanger was used for capture. Elution using arginine suppressed protein aggregation and allowed a very good resolution with purity and product-recovery was above 90%. Free PEG was removed completely. The use of hydrophobic interaction chromatography (HIC) increased purity to 98%. Increasing the size of PEG from 5 to 30 kDa increased retention on HIC and reduced it on cation exchangers. Bioactivity of PEGylated scFv was confirmed by (125)I based cell tests. Proteins modified with 5 kDa PEG showed higher bioactivity than proteins modified with larger PEGs. The combination of cation exchange and HIC provides a rational and effective basis for PEGylated scFv purification., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The mPEG-aldehyde PEGylation with two different PEG sizes and two proteins was experimentally determined with respect to yield, conversion, and selectivity. The kinetic behavior of these PEGylation reactions was simulated using a numerically solved set of differential equations. We show that the assumption of an inactivation of mPEG-aldehyde is crucial for the simulation of the overall PEGylation and that the inactivation is pH-dependent. We further demonstrate that ideal PEGylation parameters such as pH, temperature, reaction time, and protein concentration need to be chosen carefully depending on the protein and PEG size. In terms of selectivity and yield, we show that the reaction should be stopped before the highest mono-PEG concentration is reached. Moreover, room temperature and a slightly acidic pH of approximately 6 are good starting points. In conclusion, selectivity can be optimized choosing a shorter reaction time and a reduced reaction temperature.

Dynamic binding capacity (DBC) measurements of cation-exchange resins were performed with two human monoclonal antibodies. DBC showed a pH dependent maximum, which was shifted to lower pH values with increasing buffer concentrations and increasing salting-out effect of the buffer anion according to the Hofmeister series. As this downshift correlates well with zeta potential values, a measurement of the latter allows the determination of the pH value for maximum DBC under a given set of conditions. Thus, the use of zeta potential values can accelerate the purification process development and helps to understand the protein adsorption mechanism.

Tumor necrosis factor (TNF) exists in two bioactive forms, the membrane integrated form and the proteolytically derived soluble cytokine. Both forms of TNF are involved in a variety of different physiological and pathophysiological situations. Here we analyzed different human and mouse TNF-specific reagents for their ability to determine the expression of membrane-expressed TNF. The data prove some antibodies to be very useful for the analysis of transmembrane TNF expression because these antibodies distinguish between the transmembrane form of TNF and soluble TNF bound to cellular TNF receptors. In addition, we found that recombinant human TNF receptor fusion proteins are advantageous tools to analyze both human and mouse transmembrane TNF expression., (Copyright 2000 Wiley-Liss, Inc.)

The receptor-binding AB loop of recombinant human interferon-gamma (IFN-gamma) has multiple contacts with the extracellular part of the IFN-gamma receptor a chain (IFN-gammaRalpha). We explored the possible length of truncated AB loops and their conformations by molecular modelling. Deletions of two amino acids at the tip of the loop were tolerated in the model without van der Waals collisions of the AB loop with helix F. Based on these modelling results, two deletion mutants were constructed by overlap-extension PCR mutagenesis: des-(A23, D24)-IFN-gamma and des-(N25, G26)-IFN-gamma. Both mutations were tolerated by the folding pattern of recombinant human IFN-gamma, as proved by CD spectroscopy. The stability of both mutants against cosolvent-induced unfolding was equal to that of wild-type IFN-gamma. In contrast to the biophysical similarities of wild-type and mutant IFN-gamma proteins, the biological activities of both mutants dropped significantly. Antiviral activity and human leucocyte antigen (HLA)-DR induction of des-(N25, G26)-IFN-gamma was 10% that of wild-type activity. des-(A23, D24)-IFN-gamma had only 1% remaining activity. Receptor-binding experiments confirmed that both deletions had a negative influence on the affinity of recombinant human IFN-gamma to its cellular receptor. We conclude from this combined molecular modelling and mutagenesis experiments, that the reduced flexibility of the truncated AB loop abrogates the possibility of the formation of a 3(10) helix in the receptor-bound state as observed in the X-ray structure of the IFN-gammaRalpha-IFN-gamma complex.

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C mu (PKC mu) subtype, epitope tagged PKC mu was propagated in the baculovirus expression system and was purified to homogeneity. PKC mu displays high affinity phorbol ester binding (Kd=7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P2, were found to promote PKC mu kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKC mu too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKC mu.

We here describe an efficient procedure for overexpression and purification of recombinant complete human interferon-gamma (IFN-gamma) receptor (IFN-gamma-R) and its extracellular fragment employing a baculovirus (BV) expression system. Infection of Sf 158 cells with recombinant BV results in membrane expression of high affinity IFN-gamma-R (Kd 1.6 x 10(-10) M), with approximately 10(6) molecules/cell 40 h postinfection. Solubilized, affinity-purified IFN-gamma-R and a secreted extracellular domain of IFN-gamma-R were compared for ligand-binding capacity and antagonistic activity in an IFN-gamma bioassay. Our results show that the complete receptor has a 2.5-fold higher ligand affinity and a 15-fold higher IFN-gamma in vitro-neutralizing capacity in an in vitro virus protection assay as compared to the extracellular fragment. This suggests that the transmembrane and cytoplasmic domains of IFN-gamma-R contribute to stability and/or enhance formation of biologically active receptor complexes in solution.

Pseudohypoparathyroidism (PHP) is a rare disorder that might be caused by an hereditary defect in the G protein system. These membrane-bound proteins are responsible for the transduction of biological signals through the outer cell membrane. The investigation of 22 patients with PHP showed a symmetric sensorineural hearing loss in 63.6% of the subjects. In erythrocyte membrane preparations from blood samples of 15 of these patients, we measured the concentration of the stimulatory Gs protein and the inhibitory Gi protein by means of the Western blot analysis using selective antibodies against alpha-subunits of G proteins. In nine of the 15 cases (60%), we found a distinct decrease in the amount of the Gs protein with a partial preponderance of the Gi protein. These patients had a considerable symmetric sensorineural hearing loss. Pathophysiological mechanisms and the possible role of G proteins in the inner ear are discussed.

The role of the beta gamma-subunits in the interaction of G-proteins was examined with beta 1-adrenoceptors purified from turkey erythrocytes and pure beta gamma-subunits prepared from turkey erythrocytes and bovine brain. On a non-denaturing polyacrylamide gel, the mobility of beta gamma-subunits was increased when incubated with beta 1-adrenoceptor and the beta 1-adrenergic agonist 1-(-)-isoproterenol, whereas on incubation with the antagonist 1-alprenolol the mobility was unchanged. Furthermore, the beta 1-adrenoceptor was retarded on a Sephadex G-50 column equilibrated with beta gamma-subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated beta 1-adrenoceptors with isolated beta gamma-subunits of G-proteins.

The effects of botulinum C2 toxin, which ADP-ribosylates actin, were studied on the stimulated histamine release of rat peritoneal mast cells. Treatment of mast cells with botulinum C2 toxin for 4 h inhibited histamine release stimulated by compound 48/80 or MCD-peptide maximally by about 50%. A half-maximal and maximal inhibition occurred at about 30 and 450 ng/ml botulinum C2 toxin. At similar concentrations the toxin ADP-ribosylated actin in intact mast cells. Botulinum C2 toxin largely impaired the histamine release stimulated by 12-O-tetradecanoylphorbol-13-acetate but not by the ionophore A23187. The data indicate that botulinum C2 toxin partially inhibits the stimulated histamine release and suggest that actin is involved in the stimulus-secretion coupling in mast cells.