Two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis combines two different electrophoretic separating techniques in perpendicular directions to provide a much greater separation of complex protein mixtures than either of the individual procedures. Variations of the most common two-dimensional technique are described in this unit, namely isoelectrofocusing (IEF) and SDS-PAGE. This unit also includes support protocols describing pI standards and pH profile measurements, casting Immobiline gels, preparation of tissue culture cells and solid tissues for isoelectricfocusing, preparation of molecular weight standards for two-dimensional gels, and two-dimensional protein databases.

Electroblotting from polyacrylamide gels.

This unit contains procedures for electrophoretically transferring proteins onto a variety of membranes including polyvinylidene difluoride (PVDF) and nitrocellulose, and derivatized membranes. The choice of membrane type for electrotransfer is dependent on the ultimate application for the blot membrane. An alternate protocol is provided for electroblotting in semidry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents.

Two-dimensional gel electrophoresis.

While one-dimensional SDS-PAGE separates proteins on the basis of size, two-dimensional gel electrophoresis separates proteins first on the basis of isoelectric point, then on the basis of size. This method is capable of resolving 1000 to 2000 separate proteins when combined with sensitive detection methods. This unit describes methods for characterizing cell lysates by two-dimensional gel electrophoresis, including modifications for acidic and basic proteins, the use of immobilized pH gradients, and nonreducing/reducing electrophoretic separations. In addition there are support protocols for determining pH profiles of gels, casting Immobiline gels, preparing cell and tissue samples for isoelectric focusing, preparing molecular weight standards, and using two-dimensional protein databases.

Validation of a peptide mapping method for a therapeutic monoclonal antibody: what could we possibly learn about a method we have run 100 times?

Peptide mapping is a key analytical method for studying the primary structure of proteins. The sensitivity of the peptide map to even the smallest change in the covalent structure of the protein makes it a valuable 'finger-print' for identity testing and process monitoring. We recently conducted a full method validation study of an optimised reverse-phase high-performance liquid chromatography (RP-HPLC) tryptic map of a therapeutic anti-CD4 IgG1 monoclonal antibody. We have used this method routinely for over 1 year to support bioprocess development and test production lots for clinical trials. Herein we summarize the precision and ruggedness of the testing procedure and the main findings with respect to 'coverage of amino acid sequence' and limits-of-detection for various hypothetical structural variants. We also describe, in more detail, two unanticipated insights into the method gained from the validation study. The first of these is a potentially troublesome side-product arising during the reduction/alkylation step. Once the cause of this side-product was identified, it was easily prevented. We also report on subtle changes to the peptide map upon extended storage of the digest in the autosampler. These findings helped us to develop a 'robust' method for implementation in a quality control laboratory.

Protein sequence analysis using Hewlett-Packard biphasic sequencing cartridges in an applied biosystems 473A protein sequencer.

Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.g., formulated protein drugs) can be easily analyzed using the ABI sequencer. Simple modifications to the ABI sequencer to accommodate the cartridge extend its utility in the analysis of difficult samples. The ABI sequencer solvents and reagents were compatible with the HP cartridge for sequencing. Sequence information up to ten residues can be easily generated by this nonoptimized procedure, and it is sufficient for identifying proteins by database search and for preparing a DNA probe for cloning novel proteins.

An automated C-terminal sequencing method for analysis of proteins us ing an ABI 473A sequencer.

We developed a novel chemistry for C-terminal sequencing of proteins based on derivatization with acetylisothiocyanate to yield amino acid thiohydantoins (TH-AAs), and it was used for manual sequencing of biopharmaceutical products on a routine basis.This simple chemistry was automated using a ABI 473A N-terminal sequencer. All reagents (R1, trimethylsilylisothiocyanate; R3, alkaline thiocyanate for cleavage) and solvents required for sequencing were accommodated on the sequencer, which was modified to deliver liquid R2 (acetyl chloride) to the reaction vessel.The conversion flask was used for preparing the TH-AAs for analysis by online HPLC using a graphitized carbon (Hypercarb) column. Results obtained with model proteins and recombinant protein drugs suggest that at least three residues from the C terminus can be easily determined.The C-terminal heterogeneity in more than five types of recombinant immunoglobulin G was determined, and the differences in Gly-Lys ratios were consistent with changes observed in the isoelectric focusing profile of these antibodies. Because the chemistry uses only four reagents delivered to the reaction vessel and three to the conversion flask,we believe that the automated protocol can be easily adapted to any existing N-terminal sequencer.

High-yield deblocking of amino termini of recombinant immunoglobulins with pyroglutamate aminopeptidase.

For larger proteins, efficient deblocking prior to Edman sequencing is especially important to obtain quality, extended sequencing data which is limited by the stepwise accumulation of background from the random acid hydrolysis of the protein. Therefore, any portion that remains blocked contributes to the undesirable background. We report an optimized procedure for the removal of pyroglutamate (pGlu) by pyroglutamate aminopeptidase (PGAP) and demonstrate its use for the quantitative deblocking of several humanized recombinant antibodies (rIgGs). The rIgGs with blocked heavy chain provided an advantageous system in which removal of pGlu from the heavy chain was determined as a ratio of the deblocked heavy chain to the light chain in the first cycle of sequencing; i.e., the light chain was used as an internal standard. The reaction temperature, reaction time, enzyme-to-substrate ratio, denaturation, and reduction/carboxymethylation prior to digestion, and different commercial enzymes were evaluated. The optimized procedure involves reduction/carboxymethylation in guanidine buffer, buffer exchange by gel-permeation chromatography, and overnight PGAP digestion at 37 degrees C. Five different rIgGs, including one with blocked heavy and light chains, were deblocked in nearly quantitative yields using this procedure.

A pulmonary influenza virus infection in SCID mice can be cured by treatment with hemagglutinin-specific antibodies that display very low virus-neutralizing activity in vitro.

We have previously shown that a pulmonary influenza virus infection in SCID mice can be cured by treatment with monoclonal antibodies (MAbs) specific for the viral transmembrane protein hemagglutinin (HA) but not for matrix 2. Since both types of MAbs react with infected cells but only the former neutralizes the virus, it appeared that passive MAbs cured by neutralization of progeny virus rather than reaction with infected host cells. To prove this, we selected a set of four HA-specific MAbs, all of the immunoglobulin G2a isotype, which reacted well with native HA expressed on infected cells yet differed greatly (>10,000-fold) in virus neutralization (VN) activity in vitro, apparently because of differences in antibody avidity and accessibility of the respective determinants on the HA of mature virions. Since the VN activities of these MAbs in vitro were differentially enhanced by serum components, we determined their prophylactic activities in vivo and used them as measures of their actual VN activities in vivo. The comparison of therapeutic and prophylactic activities indicated that these MAbs cured the infection to a greater extent by VN activity (which was greatly enhanced in vivo) and to a lesser extent by reaction with infected host cells. Neither complement- nor NK cell-dependent mechanisms were involved in the MAb-mediated virus clearance.

Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes.

The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins. Some variability persisted even when optimized electroblotting procedures were used which reduce the sodium dodecyl sulfate (SDS) concentration in the gel and improve protein-PVDF binding. The membranes which were evaluated could be grouped as either "high retention" membranes (ProBlott, Trans-Blot, and Immobilon-PSQ) or "low retention" membranes (Immobilon-P and Westran). The high retention membranes showed higher protein recoveries under most conditions tested, especially for small proteins or peptides. These high retention membranes were also less sensitive to the exact electroblotting conditions, especially those factors which affect the amount of SDS present during either electrotransfer or direct adsorption from protein solutions. High retention PVDF membranes are therefore preferred in most cases for optimal protein or peptide recovery prior to direct sequence analysis. In contrast, low retention membranes are preferred for procedures where subsequent extraction of the proteins from the membranes is required. Even under identical conditions, substantial protein-to-protein variation for both adsorption and subsequent extraction is routinely observed for both groups of membranes, indicating that the nature of protein-PVDF interactions is more complex than simple hydrophobic interactions.

High yield electroblotting onto polyvinylidene difluoride membranes from polyacrylamide gels.

Optimal conditions of electroblotting that led to high protein recovery on polyvinylidene difluoride (PVDF) membranes were determined for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS concentrations in the gel and transfer buffer were found to be the most important factors affecting the amount of protein recovered on the PVDF membrane. The largest loss occurred during the first 10-30 min of transfer due to the relatively high initial SDS concentration in the gel. During this initial stage of transfer, most of the protein passed through the primary membrane and was partially retained on secondary and tertiary membranes. The value of presoaking gels prior to transfer to reduce the amount of SDS was evaluated by quantitating free SDS densitometrically and by correlating the reduced SDS concentration with increased electroblotting efficiency from presoaked gels. Transfer time was evaluated and no "overtransfer" was found even after very long transfer times. These results clearly indicate that proteins electroblotted onto PVDF membranes were tightly bound and could not be released by extending the transfer time. The effects of methanol and SDS concentrations on protein adsorption from solution to PVDF were also determined quantitatively. The results of this study strongly suggest that proteins fully saturated with SDS cannot bind efficiently to PVDF membranes. Since SDS is necessary for high protein mobility, the challenge in efficient electroblotting is to maintain an optimal SDS concentration which is high enough to permit effective removal from the gel and low enough to permit effective binding to the PVDF membrane. For 1.5 mm thick gels containing 0.2% SDS, presoaking the gel for 15-20 min in transfer buffer with 10% methanol prior to electroblotting provided the best recovery on the primary membrane.

Gamma-interferon-induced nerve growth factor receptors in colorectal carcinoma cell lines.

Gamma-interferon (gamma IFN) was found to induce expression of the 150,000 M(r) cell surface and the 35,000 M(r) chromatin receptors for nerve growth factor (NGF) in the SW1116 colorectal carcinoma cell line that does not express NGF receptors. In the SW707 colorectal carcinoma cell line that expresses a low level of NGF receptors, gamma IFN stimulated expression of the cell surface and the nuclear receptors. Induction of NGF receptors in SW1116 cells resulted in internalization and nuclear translocation of 125I-NGF. When NGF bound to the chromatin, ribosomal RNA synthesis was inhibited. Two-dimensional gel electrophoresis of [35S]methionine-labeled chromatin proteins indicated significant changes in chromatin protein composition in cells treated and not-treated with gamma IFN. gamma IFN effectively stimulated the expression of NGF receptors in two colorectal carcinoma cell lines, but inhibited the expression in melanoma and breast carcinoma cells. It is suggested that gamma IFN, by modulating the expression of NGF receptors may affect the NGF-dependent growth of some tumor cell lines.

[Therapeutic trial with fucidin in systemic scleroderma]. / Therapieversuch mit Fucidin bei systemischer Sklerodermie.

The signs and symptoms in systemic sclerosis were improved by systemic therapy with fucidine. In patients with untreated systemic sclerosis, asparaginic acid and glutaminic acid were increased in blood serum and asparagine and glutamine decreased. These compounds with fucidine therapy normalized.

Treatment of systemic scleroderma with fucidine with regard to some free amino acids contents before and after therapy.

In 7 patients with systemic scleroderma and acroscleroderma improvement was observed after the administration of fucidine. In the same time 4 amino acids contents, which had been abnormal prior to the therapy, normalized.