Claudia Ettori

Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with immobiline membranes

Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.

Structure-stability relationship of Immobiline chemicals for isoelectric focusing as monitored by capillary zone electrophoresis

Abstract An acrylamido buffer for isoelectric focusing in immobilized pH gradients, 1-acryloyl-4-methylpiperazine (p K = 6.85 at 25°C), was synthesized. As it is a disubstituted amide, it was thought that it would be much more resistant to alkaline hydrolysis. In reality, it degraded rapidly in 0.1 M sodium hydroxide solution at 70°C (86% in 6 h). Therefore, the stability of Immobiline buffers of p K 9.3, 8.5, 7.0 and 6.2 was investigated. Under the same hydrolysis condition, the degradation was 11%, 22%, 26% and 34%, in order of decreasing p K values. The kinetics of degradation were monitored by capillary zone electrophoresis in 0.1 M borate buffer (pH 9). The decrease of the main Immobiline peak and the appearance of its hydrolytic products, acrylic acid and a diamine, could be easily measured. The following general rules were derived: when the nitrogen engaged in the amido bond is inserted into a cyclic structure ( e.g. , 1-acryloyl-4-methylpiperazine) there is very little protection against hydrolysis, and conversely, when this nitrogen carries flexible, fairly long substituents (4–5 atoms long) much stronger shielding and protection of the amido bond can be obtained. These findings helped in designing new acrylamido derivatives strongly resistant to chemical degradation.

Capillary zone electrophoresis analysis of acrylamido buffers for isoelectric focusing in immobilized pH gradients

Abstract Studies are described using fourteen weak acrylamido acids and bases (Immobilines) as buffers and titrants for isoelectric focusing in immobilized pH gradients (IPG), plus one strongly acidic (pK 1.0) and one strongly basic (pK> 12) titrants, utilized in general to reach pH extremes in extended IPG ranges. Three fundamental properties of these buffers were evaluated: (a) resistance to alkaline hydrolysis of the amido bond; (b) hydrophilicity (so as to avoid hydrophobic interactions with the matrix during electrophoretic migration); and (c) resistance to oxidation by peroxodisulphate during the polymerization step. Capillary zone electrophoresis (CZE) was used to evaluate these properties. The acidic compounds were resolved in 100 mM acetate (pH 4) whereas the alkaline species were separated in 50 mM phosphate buffer (pH 7.7) (or pH 7.2 for the weaker compounds). All the acrylamido derivatives were detected underivatized by their absorption at 214 or 254 nm. The degradation kinetics of all compounds were monitored, after hydrolysis in 0.1 M NaOH at 70°C (up to 6 h), by CZE in 0.1 M borate buffer (pH 9). The decrease in the Immobiline peak and the appearance of its hydrolytic products (acrylic acid and a diamine in the case of the acrylamido bases) could easily be monitored and quantified in CZE by using the Gold integration system. A hydrophobicity scale was constructed by measuring the partition coefficients of the basic acrylamido derivatives in 1-octanol—water. General guidelines are given for the proper use of these chemicals and for the synthesis of additional compounds.