Fast quantitation of recombinant plasminogen activator inhibitor type 1 in bacterial lysates by micropellicular reversed-phase liquid chromatography.

A rapid reversed-phase HPLC assay is described for quantitating recombinant plasminogen activator inhibitor type 1 (PAI-1) in cultures of Escherichia coli. The assay utilized a short nonporous micropellicular C18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. The selective resolution of recombinant PAI-1 from major host proteins occurred within 1.3 min. Regeneration of initial chromatography conditions was fast and allowed successive injections every 3 min. The assay's limit of detection for recombinant PAI-1 was 0.5 microg in 5-microl injections of bacterial lysates and the assay was linear to 20 microg. The assay's apparent recovery was between 94 and 108% throughout the quantitative range. In a direct comparison to gel electrophoresis the reversed-phase assay proved superior in monitoring recombinant PAI-1 titers in cultures of E. coli.

Continuous culture study of the expression of hepatitis B surface antigen and its self-assembly into virus-like particles in Saccharomyces cerevisiae.

We have studied the growth rate dependence of hepatitis B surface antigen (HBsAg) p24(s) monomer and lipoprotein particle synthesis produced in Saccharomyces cerevisiae using galactose-limited continuous culture. The hepatitis B virus S gene, which encodes the p24(s) monomer, is transcribed under the control of the GAL 10p on a chimeric 2-microm plasmid harbored in a haploid yeast strain. Monomers autonomously form lipoprotein aggregates (particles) in vivo using only host-cell-derived components. Steady states were evaluated in a range from 0.015 h(-1) to washout (0.143 h(-1)). Both p24(s) monomer and HBsAg particle levels, at steady state, varied in an inverse linear manner with growth rate. A consistent excess of total p24(s) monomer to HBsAg particle, estimated at five- to tenfold by mass, was found at all dilution rates. The average copy number of the 2-microm plasmid (carrying LEU2 selection) remained constant at 200 copies per cell from washout to 0.035 h(-1). Surprisingly, the average copy number was undetectable at the lowest dilution rate tested (0.015 h(-1)), even though HBsAg expression was maximal. Total p24(s) monomer and HBsAg particle values ranged twofold over this dilution rate range. No differences in the trends for HBsAg expression and average copy number could be detected past the critical dilution rate where aerobic fermentation of galactose and ethanol overflow were observed. HBsAg expression in continuous culture was stable for at least 40 generations at 0.100 h(-1).

Assay for recombinant hepatitis B surface antigen using reversed-phase high-performance liquid chromatography.

Several HPLC assays are reported for monitoring the mass of recombinant hepatitis B surface antigen (rHBsAg) in yeast cell lysates. The assays utilized either a polymeric resin column containing a phenyl ligate or a silica-based octadecyl micropellicular column. Prior to chromatography on the polymeric column, the samples were derivatized with the thiol-specific fluorescent probe monobromobimane to discriminate the rHBsAg from nonfluorescent cellular components. Using a dual gradient of acetic acid and acetonitrile the derivatized rHBsAg eluted with a retention time equal to 17 min. Chromatography on the micropellicular column did not require prederivatization and utilized an isopropanol gradient with increasing amounts of acetonitrile. Operating this column at elevated temperature with a high flow rate resolved the rHBsAg from yeast components within 5 min and allowed a new sample injection every 10 min. All the assays displayed useful linear ranges for analyzing rHBsAg in cell lysates and had detection limits for rHBsAg between 10 and 50 ng per injection.

Quantitative electrophoretic analysis of proteins labeled with monobromobimane.

Protein cysteine residues were labeled with the thiol-specific fluorescent probe monobromobimane prior to electrophoresis. Labeling was rapid and complete under the conditions employed. Scanning the negative films of photographed gels with a laser densitometer provided quantitation. Analysis of five different cysteine-containing proteins demonstrated that the fluorescence was dependent on the total number of cysteine residues present for each protein and independent of the protein species. The method allowed the construction of two nomograms for the universal quantitative analysis of cysteine-containing proteins. One nomogram calculated the limit of detection for each protein based on the protein's molecular mass, the protein's molar ratio of cysteine, and a nominal detection limit of 10 pmol cysteine. The second nomogram determined the quantity of the class 4 protein in outer membrane extracts of Neisseria meningitidis from the protein's molecular mass, its molar ratio of cysteine, and its total cysteine content. Additional applications for the analysis of protein cysteine and cystine content are discussed.

Identification of an Escherichia coli protein impurity in preparations of a recombinant pharmaceutical.

A host-cell protein impurity found in preparations of recombinant human acidic fibroblast growth factor (aFGF) was identified. Samples of aFGF examined by western blot analysis employing antiserum raised against an Escherichia coli cell lysate contained an immunoreactive protein with a molecular weight of approximately 26,000. The impurity was chromatographically isolated and the N-terminal sequence was determined. Comparing the sequence to a protein database provisionally identified the isolated impurity as the S3 ribosomal protein of E. coli. Monoclonal antibodies recognizing three separate epitopes of S3 confirmed the identity of the impurity in western blots of aFGF samples. The monoclonal antibodies were also used to estimate S3 levels in various preparations of aFGF.

Characterization of a full-length, active-site mutant of diphtheria toxin.

A full-length recombinant mutant of diphtheria toxin containing serine in place of a crucial active-site glutamate has been purified and characterized. The serine substitution caused a minor structural alteration in the toxin as measured by trypsinolysis. ADP-ribosyltransferase activity and cytotoxicity of the mutant were both decreased by approximately 500-fold. A similar reduction in cytotoxicity was found when the enzymic fragments of both the wild-type and mutant toxins were introduced into the cytosol of fibroblasts by osmotically lysing pinosomes. The mutation did not alter the binding of the toxin to cell surface receptors and had no apparent effect on membrane translocation. The results suggest that the decreased cytotoxicity of the mutant is solely due to the reduced ADP-ribosyltransferase activity.

pH-dependent insertion of proteins into membranes: B-chain mutation of diphtheria toxin that inhibits membrane translocation, Glu-349----Lys.

To investigate how diphtheria toxin (DT) undergoes pH-dependent membrane translocation in mammalian cells, we have isolated and characterized mutants of the toxin that are defective in acidic-pH-dependent killing of Escherichia coli. Cloned DT secreted to the periplasm of E. coli kills the bacteria under acidic conditions (near pH 5.0) by inserting into and permeabilizing the inner membrane (a mechanism independent of the toxin's ADP-ribosylation activity). Mutant forms of DT with reduced lethality for E. coli were selected by plating the bacteria under acidic conditions. CRM503, one of the full-length mutants selected by this protocol, also showed diminished cytotoxicity for mammalian cells. We traced the altered cytotoxicity of CRM503 to a Glu-349----Lys mutation (E349K), one of three point mutations, within the B fragment. The E349K mutation alone inhibited cytotoxicity and membrane translocation in mammalian cells and lethality for E. coli but did not affect enzymic activity or receptor binding. The recently determined crystallographic model of DT shows that Glu-349 resides within a short loop connecting two long hydrophobic alpha-helices of the translocation domain. Protonation of Glu-349 and two other nearby acidic residues, Asp-352 and Glu-362, may enable these helices to undergo membrane insertion and the intervening loop to be transferred to the opposite face of the bilayer. The E349K mutation introduces a positive charge at this site, which would be expected to inhibit membrane insertion and the insertion-dependent activities of DT. These results suggest that protonation of Glu-349 and nearby acidic residues may be important in triggering the translocation step of toxin action.

Two pathways of transferrin recycling evident in a variant of mouse LMTK- cells.

We describe here the properties of a variant cell line, termed AF192, selected by exposing mouse LMTK- cells to a cytotoxic form of transferrin prepared by conjugating transferrin to diphtheria toxin. AF192 cells were mildly resistant to the transferrin-diphtheria toxin conjugate and were cross-resistant to the protein toxins modeccin, abrin, ricin, and Pseudomonas aeruginosa exotoxin A. AF192 cells had an aberrant transferrin cycle characterized by an approximately 50% reduction in the rate of iron uptake from diferric transferrin, an approximately 25% reduction in the number of surface transferrin receptors, and a time course for transferrin recycling that resolved into two apparent first-order rate processes. The aberrant transferrin cycle was not the result of a failure of endocytosed transferrin to discharge iron; rather, part, but not all, of the transferrin taken up by AF192 cells was diverted to an intracellular site from which it was recycled very slowly.

Characterization of a transferrin-diphtheria toxin conjugate.

We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis.

Identification of a cold-sensitive step in the mechanism of modeccin action.

Modeccin is a toxic lectin that arrests protein synthesis in mammalian cells by catalytically inactivating 60 S ribosomes. To interact with 60 S ribosomes, the catalytic subunit of modeccin must pass through a membrane and enter the cytosol. Two known steps in the mechanism of modeccin action are the receptor-mediated internalization of the toxin into vesicles and a second step that requires a low pH within the vesicles. We report here another step required for modeccin to arrest protein synthesis, identified because this step was blocked at 15 degrees C. Modeccin traveling from cell surface receptors to the cytosol at 37 degrees C passed the low pH step within vesicles in a minimum time of 15 min after endocytosis and reached the cold-sensitive step 15 min later. There was no effect on protein synthesis until about 45 min after modeccin had passed the cold-sensitive step, suggesting that the toxin was still within vesicles at the time of the cold-sensitive event. The low temperature at which modeccin failed to reach the cytosol correlated with an apparent low temperature block in the transfer of endocytosed modeccin to lysosomes. The possibility is discussed that modeccin does not penetrate to the cytosol directly from endocytic vesicles.