Ion exchange chromatography of proteins and clearance of aggregates.

Clearance of product related aggregates in therapeutic proteins is a major focus of purification process development. A typical purification process will have one or two chromatographic steps that remove these product related aggregates to an acceptable level. Both cation exchange and anion exchange chromatography can provide robust clearance of aggregates. The primary factors that are critical for aggregate clearance are: resin chemistry, binding and elution condition, peak collection and column load factor. This review covers how these factors can be optimized to increase the effectiveness of ion exchange chromatography in removing aggregates.

Prussian blue modified glassy carbon electrodes-study on operational stability and its application as a sucrose biosensor.

Stabilisation of electrochemically deposited Prussian blue (PB) films on glassy carbon (GC) electrodes has been investigated and an enhancement in the stability of the PB films is reported if the electrodes are treated with tetrabutylammonium toluene-4-sulfonate (TTS) in the electrochemical activation step following the electrodeposition. A multi-enzyme PB based biosensor for sucrose detection was made in order to demonstrate that PB films can be coupled with an oxidase system. A tri-enzyme system, comprising glucose oxidase, mutarotase and invertase, was crosslinked with glutaraldehyde and bovine albumin serum on the PB modified glassy carbon electrode. The deposited PB operated as an electrocatalyst for electrochemical reduction of hydrogen peroxide, the final product of the enzyme reaction sequence. The electrochemical response was studied using flow injection analysis for the determination of sucrose, glucose and H(2)O(2). The optimal concentrations of the immobilisation mixture was standardised as 8U of glucose oxidase, 8U of mutarotase, 16U of invertase, 0.5% glutaraldehyde (0.025mul) and 0.5% BSA (0.025mg) in a final volume of 5mul applied at the electrode surface (0.066cm(2)). The biosensor exhibited a linear response for sucrose (4-800muM), glucose (2-800muM) and H(2)O(2) (1-800muM) and the detection limit was 4.5, 1.5 and 0.5muM for sucrose, glucose and H(2)O(2), respectively. The sample throughput was ca. 60 samples h(-1). An increase in the operational and storage stability of the sucrose biosensor was also noted when the PB modified electrodes were conditioned in phosphate buffer containing 0.05M TTS during the preparation of the PB films.

Liquid chromatographic determination of total and beta-N-oxalyl-L-alpha,beta-diaminopropionic acid in lathyrus sativus seeds using both refractive index and bioelectrochemical detection.

A further improved chromatographic method for the simultaneous determination of the total amount of ODAP, selectively the amount of its neurotoxic form, beta-ODAP, and free L-glutamate in raw Lathyrus sativus (grass pea) seed samples is described using post-column refractive index in combination with bioelectrochemical detection. The biosensor is based on crosslinking horseradish peroxidase (HRP) and an Os-containing mediating polymer with poly(ethyleneglycol)(400) diglycidyl ether (PEGDGE), forming an inner hydrogel layer and then immobilising L-glutamate oxidase (GlOx) as an outer layer on top of a graphite electrode. Addition of polyethylenimine (PEI) to the hydrogel is believed to have sensitivity and stability enhancing effect on the biosensor. The double-layer approach in the biosensor construction avoided direct electrical wiring of GlOx and resulted in a higher sensitivity of 4.6 mA/M cm2 with respect to beta-ODAP and a wider linear range (1-250 microM) for both L-glutamate and beta-ODAP when compared with a single-layer approach where GlOx, HRP, and Os-polymer are crosslinked together. The limit of detection for the chromatographic-biosensor system was found to be 2 microM with respect to beta-ODAP and 0.7 microM with respect to L-glutamate. The refractive index detection on-line with the biosensor enabled full control of the chromatographic system for the determination of the total amount of ODAP, selectively the amount of beta-ODAP and L-glutamate. Ten grass pea samples have been collected from Lathyrism prone areas of Ethiopia to test the applicability of the presently developed analytical system for real sample analysis. The toxin levels of grass pea collections were determined in an aqueous extracts and ranged from 0.52 to 0.76%, dry mass basis. Comparison of results of an established spectrophotometric assay and that of the present system has shown an extraordinary degree of agreement as revealed by parallel "t" test (90% confidence limit). The present system has operational stability of more than 50 h. Analysis time per sample is 10 min after extraction for 90 min.

Isolation and characterization of an alpha-macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment.

A proteinase inhibitor with M(r) 697000 and 20.3% (w/w) carbohydrate was isolated from the haemolymph of the snail Helix pomatia and characterized. It was shown to have a tetrameric structure with subunits disulphide linked by two. It inhibited the activity of several types of proteinases against large substrates but not that of trypsin against N-alpha-benzoyl-DL-arginine-4-nitroanilide. This indicated a nonspecific and steric hindrance mode of inhibition. The ratio of trypsin molecules inactivated per inhibitor amounted to 1.5. This interaction led to a cleavage of the subunits into two equal fragments and to a slow to fast conformational change of the whole molecules. Experiments with 125I-labelled trypsin indicated that the proteinase had become covalently linked to one of the fragments. Heating of the inhibitor led to autolytic cleavage products but not when methylamine treated. Thiol titration after trypsin or methylamine treatment indicated the presence of one thiol ester bond per subunit. These facts are all indicative of an alpha-macroglobulin type of inhibitor. However, unlike for most of them the methylamine treatment did not induce a conformational change nor suppress its proteinase inhibitory activity. Moreover, invertebrate alpha-macroglobulins are mostly dimeric in structure but tetramers likewise do occur in Biomphalaria glabrata.

The C terminus of sprouty is important for modulation of cellular migration and proliferation.

The Drosophila Sprouty (SPRY) protein has been shown to inhibit the actions of epidermal growth factor and fibroblast growth factor. However, the role of mammalian SPRY proteins has not been clearly elucidated. We postulated that human Sprouty2 (hSPRY2) is an inhibitor of cellular migration and proliferation. Indeed, using stably transfected HeLa cells, which expressed hemagglutinin (HA)-tagged hSPRY2 or hSPRY2 tagged at the C terminus with red fluorescent protein, we demonstrated that hSPRY2 inhibits the migration of cells in response to serum, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor. Additionally, hSPRY2 also inhibited the growth of HeLa cells in response to serum. Previously, two C-terminal domains on hSPRY2, which are necessary for its colocalization with microtubules (residues 123-177) or translocation to membrane ruffles (residues 178-194), have been identified (Lim, J., Wong, E. S., Ong, S. H., Yusoff, P., Low, B. C., and Guy, G. R. (2000) J. Biol. Chem. 275, 32837-32845). Therefore, using TAT-tagged hSPRY2 and its mutants, we determined the role of these two C-terminal domains in the inhibition of cell migration and proliferation. Our data show that the deletion of either of these two regions in hSPRY2 abrogates its ability to modulate cell migration in response to different growth factors and proliferation in response to serum. Therefore, we conclude that hSPRY2 inhibits the actions of a number of growth factors, and its C terminus, which is homologous among various SPRY isoforms, is important in mediating its biological activity.

Cysteine 3 is not the site of in vitro palmitoylation on G(salpha).

Several studies have examined the role of palmitoylation of G protein alpha subunits by nonenzymatic in vitro acylation using palmitoyl-CoA. Here, we investigated nonenzymatic palmitoylation of purified G(salpha) in vitro. GDP-bound G(salpha) was stoichiometrically autoacylated on cysteine residue(s) with micromolar concentrations of palmitoyl-CoA. The acylation led to a complete loss of steady-state GTPase activity and GTPgammaS binding to G(salpha). Mutation of Cys 3 to Ala in G(salpha) did not prevent either palmitoylation or its consequent functional alterations. However, stoichiometric palmitoylation of His(6)-G(salpha) did not alter its GTPase activity or GTPgammaS binding. Isoelectric focusing of tryptic peptides from autoacylated wild type, His(6)-tagged, and C3A mutant of G(salpha) showed that Cys 160 is the site of in vitro palmitoylation. Therefore, we conclude that in vitro palmitoylation of G(salpha) occurs on Cys 160 and this modification decreases the ability of the protein to exchange GTP for GDP; N-terminus elongation of G(salpha) prevents this latter effect without altering palmitoylation.

Regions on adenylyl cyclase that are necessary for inhibition of activity by beta gamma and G(ialpha) subunits of heterotrimeric G proteins.

The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with beta gamma subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G(salpha) (G(salpha)*) or Ca(2+)/calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by beta gamma subunits, the presence of both of these regions formed another beta gamma interaction site that was sufficient to observe G(salpha)*- or Ca(2+)/CaM-stimulated activity. Inhibition of AC activity attributable to interaction of beta gamma subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of beta gamma on various effectors. Moreover, the C1 region of ACI was sufficient to observe G(ialpha1)-elicited inhibition of Ca(2+)/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G(ialpha1), the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of G(ialpha1) are mediated on the C1 regions of both ACI and ACV. The effects of beta gamma on ACI can be mediated by interactions with the C1a region and a beta gamma interacting site formed by the C1b and C2 domains of this enzyme.

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.