Staffan Birnbaum

Formation of proinsulin by immobilized Bacillus subtilis.

There has been an increasing interest in the use of immobilized cells for the production of pharmaceuticals as well as for products such as high fructose syrup or ethanol1. Some of these compounds are now produced on an industrial scale2 whereby the cells are used in a resting or growing state or in a nonviable form as natural carriers of the enzyme(s) involved in the synthesis. The advantages of immobilized cell technology should also apply to microorganisms modified by recombinant DNA techniques to produce a variety of eukaryotic proteins such as hormones. We describe here the properties of immobilized Bacillus subtilis cells carrying plasmids encoding rat proinsulin. Cell proliferation normally coupled to DNA replication is undesirable in immobilized cell systems as ‘clogging’ of the system occurs due to cells growing outside the beads. Therefore, different ways were investigated to inhibit cell division while allowing continued protein synthesis. We found that the addition of certain antibiotics in the growth medium, such as novobiocin which inhibits DNA replication3, fulfills these requirements, allowing proinsulin synthesis and excretion to take place over a period of several days.

Hydrophobic Interaction Capillary Electrochromatography of Protein Mutants. Use of Lipid-Based Liquid Crystalline Nanoparticles as Pseudostationary Phase

Nanoparticle-based hydrophobic interaction-capillary electrochromatography was utilized for separation of proteins with similar mass-to-charge ratio at neutral pH without organic modifier. Lipid-based liquid crystalline nanoparticles were prepared and used as pseudostationary phase,benefiting from their high biocompatibility, ease of preparation,and suspension stability at high concentrations.Use of laser-induced fluorescence enabled detection at high nanoparticle concentrations. Green fluorescent protein(GFP) and mutants of GFP harboring single or double amino acid substitutions with the same charge were separated in the described system but not in conventional capillary electrophoresis. Separation was achieved by increasing the salt concentration to promote hydrophobic interactions by shielding of the repulsive electrostatic interactions. In addition, the method was adapted to a capillary with an effective length of 6.7 cm, enabling fast separations and future applications on chip.

Separation of oxidized and deamidated human growth hormone variants by isocratic reversed-phase high-performance liquid chromatography

Reversed-phase high-performance liquid chromatography (RP-HPLC) was utilized for the separation of recombinant human growth hormone (hGH) variants on a C18 silica column at 55 degrees C using an isocratic mobile phase which contained 27% 1-propanol in a 25 mM potassium phosphate buffer, pH 6.5. Three of the obtained peaks were characterized by tryptic mapping and mass spectrometry; two of the peaks were found to contain oxidized hGH (dioxy Met14/Met125 and Met125 sulfoxide) while the third contained a deamidated form (Asn149-->Asp149 or Asn152-->Asp152). Compared to the European Pharmacopoeia RP-HPLC method of hGH analysis, this new method gives two additional peaks and a 50% reduction in the analysis time.

Nanoparticle-based pseudostationary phases in CEC: A breakthrough in protein analysis?

This review focuses on major trends in nanoparticle‐based pseudostationary phase (PSP) CEC since the publication of our previous reviews within nanoparticle‐based CEC [Nilsson, C., et al., Electrophoresis 2006, 27, 76‐83; Nilsson, C., et al., J. Chromatogr. A 2007, 1168, 212‐224.]. Special attention is given to the development toward protein analysis, which is driven by the strong emergence of protein drug development in the pharmaceutical industry. Furthermore, we discuss the development in coupling different detection techniques with nanoparticle‐based PSP CEC, which were originally predicted to be particularly cumbersome. However, at present, direct UV, LIF and ESI‐MS have been used without any severe complications. Different types of nanoparticles used as PSP during the period include gold nanoparticles, carbon nanostructures and lipid‐based nanoparticles. New materials (for example, different types of carbon nanostructures and self assembled lipid‐based nanostructures) are a strong driving force for development in separation science. Finally, future trends in nanoparticles‐based CEC are envisioned.

Nanoparticle‐based capillary electroseparation of proteins in polymer capillaries under physiological conditions

Totally porous lipid‐based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas®, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle‐based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single‐amino‐acid‐substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one‐step procedure based on self‐assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid‐based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary‐based method facilitates future electrochromatography of proteins on polymer‐based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development.

Cationic and anionic lipid-based nanoparticles in CEC for protein separation.

The development of new separation techniques is an important task in protein science. Herein, we describe how anionic and cationic lipid‐based liquid crystalline nanoparticles can be used for protein separation. The potential of the suggested separation methods is demonstrated on green fluorescent protein (GFP) samples for future use on more complex samples. Three different CEC‐LIF approaches for protein separation are described. (i) GFP and GFP N212Y, which are equally charged, were separated with high resolution by using anionic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (ii) High efficiency (800 000 plates/m) and peak capacity were demonstrated separating GFP samples from Escherichia coli with cationic nanoparticles suspended in the electrolyte and adsorbed to the capillary wall. (iii) Three single amino‐acid‐substituted GFP variants were separated with high resolution using an approach based on a physical attached double‐layer coating of cationic and anionic nanoparticles combined with anionic lipid nanoparticles suspended in the electrolyte. The soft and porous lipid‐based nanoparticles were synthesized by a one‐step procedure based on the self‐assembly of lipids, and were biocompatible with a large surface‐to‐volume ratio. The methodology is still under development and the optimization of the nanoparticle chemistry and separation conditions can further improve the separation system. In contrast to conventional LC, a new interaction phase is introduced for every analysis, which minimizes carry‐over and time‐consuming column regeneration.

CHARACTERIZATION OF PERIPLASMIC ESCHERICHIA COLI PROTEIN EXPRESSION AT HIGH CELL DENSITIES

We have used two‐dimensional electrophoresis (2‐DE) to analyze changes in protein expression profiles during a microbial cultivation process on an industrial scale. An Escherichia coli strain W3110 containing the gene for recombinant human growth hormone production was used. Samples were taken at time intervals ranging from fast to slow growth rate (late growth phase at high cell density/starvation) and 2‐DE analysis combined with image analysis using the PDQuest software showed significant alterations in expression levels of a number of proteins. Twenty‐four protein spots were identified using a combination of matching with SWISS‐2DPAGE E. coli map, N‐terminal sequence analysis and mass spectrometry matrix‐assisted laser desorption/ionization (MALDI). Two of the most abundant proteins expressed at late growth phase (pI 5.4/28 kDa and pI 5.5/28 kDa) were subjected to N‐terminal sequence analysis after electrotransfer of the proteins from a preparative 2‐DE gel to polyvinylidene difluoride (PVDF) membrane. Sequence tags of five amino acids in combination with approximate pI and Mr identified both proteins as deoxyribose phosphate aldolase (gene name deoC). In addition, both spots were subjected to tryptic in‐gel digestion and analyzed using MALDI. Peptide mass fingerprints from both spots showed similar MALDI spectra and 10 of 10 tryptic fragments confirmed the identity as deoC. The identification of the acidic variant of deoC on 2‐DE gels and the observation of this variant as induced during late growth phase is novel.

[57] Production of proinsulin by entrapped bacteria with control of cell division by inhibitors of DNA synthesis

Publisher Summary This chapter describes the production of proinsulin by entrapped bacteria with control of cell division by inhibitors of DNA synthesis. With the development of recombinant DNA technology, novel microorganisms have become available, which produce a variety of eukaryotic proteins such as hormones. Currently, most genetically engineered bacteria are batch-fermented in a nonimmobilized form to produce the desired protein. However, owing to the inherent advantages of cell immobilization it is on many occasions worthwhile investigating the potential of combining immobilized cell technology and gene technology. The production of polypeptides by microorganisms requires that the transcriptional and translational machineries of the cell are functional. In the past, this has necessitated cell reproduction as well. Such reproduction often causes problems in fermentation processes based on immobilized cells as proliferating cells often clog the matrices in which they are embedded, thus impeding the flow of nutrients and eventually stopping the process. Additionally, because of cell division, the cells become dislodged from the support and contaminate the product.

[30] Automated TELISA procedure for process monitoring

Publisher Summary The synthesis of valuable proteins by recombinant prokaryotic or eukaryotic cells in the laboratory as well as in industrial-scale biotechnological processes requires rapid, sensitive, specific, and automated means of determination for the optimization and control of the production system. This can be achieved by combining the enzyme-linked immunosorbent assay (ELISA) technique, with the enzyme thermistor as detector for the enzyme activity to form a thermometric enzyme-linked immunosorbent assay (TELISA). This chapter illustrates an automated TELISA system for the analysis of insulin that is used to monitor the production of human proinsulin in fermentation broth by Escherichia coli . The experimental design of the analytical apparatus is presented. The autosampler contains three test tubes for each fermentation sample analyzed. The first test tube contains the fermentation supernatant or the standard sample mixed with an equal volume of the insulin-peroxidase conjugate. The second tube contains the substrate for the peroxidase reaction. The third tube contains 0.2 M glycine–HCl, pH 2.2.