Replacement of immobilised cell bioreactors by smaller immobilised enzyme bioreactors: unique-outcome predictability for cytochromes P450 isoforms?

Both immobilized enzymes (IME) and immobilized cells (IMC) are acceptable as the biocatalysts essential for the attainment of rapid rates of bioconversion in bioreactors. IMC can display higher than expected cellular permeability whilst IME can exhibit high catalytic constant (k_cat/K_m) despite limitations on substrate utilisation due to an unstired diffusion layer of solvent. Scale-down switching from IMC to IME involves the replacement of high-volume biotechnology by low-volume biotechnology, sometimes using IME mimics in partially non-aqueous solvent systems. Highly purified IME systems covalently immobilised to particles of, for instance, microcrystalline cellulose or porous glass, can retain both the hydrophilic and hydrophobic intermediate products in situ of the chosen sequence of enzyme reactions. These bioconversions, therefore, are as efficient as those with IMC where enzymes are often particle- or membrane-bound so that even hydrophilic intermediates are not released rapidly into solution. This mimicry of in vivo biosynthetic pathways that are compartmentalised in vivo (e.g. of lysosomes, mitochondria and endoplasmic reticulum) can replace larger IMC by IME especially in application of up to 2700 cytochromes P450 isoforms in bioprocessing. In silico investigation of appropriate model IME systems, in comparison with IMC systems, will be needed to define the optimal bioreactor configuration and parameters of operation, such as pH, T and oxygen mass transfer rate (OTR). The application solely of hazop (applied hazard and operability concepts) may, nevertheless, not be recommended to replace fully the in silico and real-lab pilot-scale and scale studies. Here, food-safe bioprocessing has to be achieved without incorporation of recognised biohazards; especially in the form of unacceptable levels of toxic metals that promote a risk-analysis uncertainty.     

Evaluation of an alternative IMS dissociation procedure for use with Method 1622: detection of Cryptosporidium in water

U.S. EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 degrees C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.     

Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation

A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (approximately 200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion MS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKC(alpha), one within PKC(delta), and two within PKC(zeta)). Internal calibration improved the mass accuracy for LC MS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.     

Maintaining connexin43 gap junctional communication in v-Src cells does not alter growth properties associated with the transformed phenotype

Loss of connexin expression and/or gap junctional communication (GJC) has been correlated with increased rates of cell growth in tumor cells compared to their normal communication-competent counterparts. Conversely, reduced rates of cell growth have been observed in tumor cells that are induced to express exogenous connexins and re-establish GJC. It is not clear how this putative growth-suppressive effect of the connexin proteins is mediated and some data has suggested that this function may be independent of GJC. In mammalian cells that express v-Src, connexin43 (Cx43) is phosphorylated on Tyr247 and Tyr265 and this results in a dramatic disruption of GJC. Cells that express a Cx43 mutant with phenylalanine mutations at these tyrosine sites form functional gap junctions that, unlike junctions formed by wild type Cx43, remain functional in cells that co-express v-Src. These cells still appear transformed; however, it is not known whether their ability to maintain GJC prevents the loss of growth restraints that confine "normal" cells, such as the inability to grow in an anchorage-independent manner or to form foci. In these studies, we have examined some of the growth properties of cells with Cx43 gap junctions that remain communication-competent in the presence of the co-expressed v-Src oncoprotein.     

Suppression of high-fidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha,a chemical inhibitor of p53

We investigated the effect of pifithrin-alpha (PFTalpha), a chemical inhibitor of p53, on DNA double-strand break (DSB) repair in mammalian chromosomes. Thymidine kinase-deficient mouse fibroblasts were stably transfected with DNA substrates containing one or two recognition sites for yeast endonuclease I-SceI embedded within a herpes simplex virus thymidine kinase gene. Genomic DSBs were induced by introducing an I-SceI expression plasmid into cells in the presence or absence of 20 microM PFTalpha. From cells containing the DNA substrate with a single I-SceI site we recovered low-fidelity nonhomologous end-joining (NHEJ) events in which one or more nucleotides were deleted or inserted at the DSB. From cells containing the substrate with two I-SceI sites we recovered high-fidelity DNA end-joining (precise ligation (PL)) events. We found that treatment of cells with PFTalpha caused a 5-10-fold decrease in recovery of PL but decreased recovery of NHEJ by less than two-fold. Deletion sizes associated with NHEJ were unaffected by treatment with PFTalpha. Our work suggests the possibility that p53 facilitates high-fidelity DSB repair while playing little or no role in mutagenic NHEJ.     

Proteome analysis of the rat cornea during angiogenesis

Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. However, the study of this complex process is often hampered by the lack of a suitable cell-based model and the tools to study the biochemical events that lead to new blood vessel growth. The most widely accepted model for angiogenesis is the in vivo rat corneal model. In this model, the cornea, which is normally an avascular tissue, is stimulated to undergo angiogenesis in response to silver nitrate cauterization or to the implantation of an exogenous angiogenic agent. The physical changes associated with the new vessel growth can be readily monitored visually, but the regulated biochemical events that result in the growth and remodeling of the new vessels are much more challenging to decipher. In this report, a proteomics approach is evaluated for its utility in deciphering the biochemical events during a time course of angiogenic stimulation. At various time points post-silver nitrate cautery, corneas were harvested, solubilized, and analyzed by two-dimensional gel electrophoresis. Protein expression profiles at the various stages of angiogenesis were compared to those of control corneas. One hundred and eleven differentially-expressed proteins were identified by either matrix-assisted laser desorption/ionization-time of flight mass spectrometry or liquid chromatography-coupled electrospray ionization tandem mass spectrometry. Many of the proteins up-regulated during the angiogenesis process were identified as blood-related proteins, thus validating the development of functional blood vessels in the normally avascular tissue of the cornea. Furthermore, detection of differentially-regulated proteins in cauterized versus control tissue clearly validated the utility of a proteomics approach to study this model of angiogenesis. However, in order to get at the key regulatory proteins in the angiogenesis process, it is clear that additional scale-up and enrichment approaches will be required.     

Stability of recombinant protein production in the GS-NS0 expression system is unaffected by cryopreservation

Mammalian cells form a very important part of the repertoire of production systems available to scientists involved in the production of recombinant proteins. During the production of therapeutic proteins it is vital for regulatory approval of products that no phenotypic or genetic changes are observed in the cell line or product. As part of the generation and development of therapeutic protein production, cell lines have to be frozen at various stages to create cell banks. If cryopreservation and revival of frozen stocks were to give rise to any phenotypic changes in the cells, this would again be detrimental to the further development of that particular cell line. This study uses one of the most industrially important expression systems, the GS-NS0 expression system, to examine the effect of cryopreservation on the growth and productivity profile of cell lines that exhibit differential degrees of stability during prolonged (production) culture periods. Results show that cryopreservation and revival procedures do not alter the stability characteristics of cell lines. This type of information is of great value in definition of protocols for cell line development.     

Cloning,over-expression,purification, and characterisation of N-acetylneuraminate synthase from Streptococcus agalactiae

N-acetylneuraminate synthase (NeuAc-synthase; E.C. 4.1.3.19) is one of the two enzymes responsible for sialic acid (N-acetylneuraminic acid) synthesis in bacteria. Potential genes encoding NeuAc synthase in Streptococcus agalactiae and Bacillus subtilis were identified from a BLAST search of the EMBL/GenBank/DDBJ database using the E. coli neuB gene sequence as a probe and the genes cloned and expressed at high level in Escherichia coli. The neuB gene of S. agalactiae was shown to encode an active NeuAc synthase, whereas the spsE gene product from B. subtilis did not have this activity. Expression of the native S. agalactiae neuB gene product enzyme in E. coli resulted in a product that was prone to proteolysis during purification so the protein was tagged with a hexa-histidine tag at its N-terminus and the enzyme was rapidly purified to homogeneity by ammonium sulphate fractionation and Ni-chelating affinity chromatography in two steps. Measurement of the subunit molecular mass by electrospray ionisation mass spectrometry (M(r) = 38, 987 +/- 3) and of the native molecular mass by gel filtration chromatography (M(r) = 78,000) clearly demonstrated that the enzyme is dimeric. The effects of EDTA, temperature, and pH on the activity of the S. agalactiae NeuAc synthase were examined. Enzyme activity was maximal at pH 7 and was dependent on the presence of metal ions such as Mg(2+), Mn(2+) or Co(2+). The purified enzyme was inhibited by the reagent phenylglyoxal and the substrates N-acetyl mannosamine or phosphoenol pyruvate afforded protection against this inhibition, suggesting that one or more arginine residues are involved in substrate recognition and binding. The ease of expression and the properties of the enzyme should now permit a thorough study of the specificity of the enzyme and provide the prerequisites for attempts to alter this specificity by directed evolution for the production of novel sialic acid analogues.     

Overexpression,purification, and structural analysis of the hydrophobic E5 protein from human papillomavirus type 16

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.     

Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy

Atomic force microscopy was used in ambient conditions to directly image dense and sparse monolayers of bovine fetal epiphyseal and mature nasal cartilage aggrecan macromolecules adsorbed on mica substrates. Distinct resolution of the non-glycosylated N-terminal region from the glycosaminoglycan (GAG) brush of individual aggrecan monomers was achieved, as well as nanometer-scale resolution of individual GAG chain conformation and spacing. Fetal aggrecan core protein trace length (398+/-57 nm) and end-to-end length (257+/-87 nm) were both larger than that of mature aggrecan (352+/-88 and 226+/-81 nm, respectively). Similarly, fetal aggrecan GAG chain trace length (41+/-7 nm) and end-to-end (32+/-8 nm) length were both larger than that of mature aggrecan GAG (32+/-5 and 26+/-7 nm, respectively). GAG-GAG spacing along the core protein was significantly smaller in fetal compared to mature aggrecan (3.2+/-0.8 and 4.4+/-1.2nm, respectively). Together, these differences between the two aggrecan types were likely responsible for the greater persistence length of the fetal aggrecan (110 nm) compared to mature aggrecan (82 nm) calculated using the worm-like chain model. Measured dimensions and polymer statistical analyses were used in conjunction with the results of Western analyses, chromatographic, and carbohydrate electrophoresis measurements to better understand the dependence of aggrecan structure and properties on its constituent GAG chains.