Empty capsids in column-purified recombinant adenovirus preparations.

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

Carboxyalkylated histidine is a pH-dependent product of pegylation with SC-PEG.

PURPOSE: Pegylation of therapeutic protein usually results in a mixture of monopegylated proteins with differing sites of modification. With rh-interferon-alpha2A pegylation, we have found that this heterogeneity includes two classes of pegylation site chemistry, the relative proportions of which can be adjusted by reaction pH. METHODS: The effect of pegylation reaction pH on the relative proportion of three peaks produced was investigated. Products were purified and characterized by peptide mapping, chemical stability to neutral hydroxylamine, and biologic activity. RESULTS: Reactions at basic pH levels produced a mixture of products pegylated at lysine residues as has been observed elsewhere. However, the dominant product of reactions at mildly acidic levels of pH showed distinct chemistry and higher cytopathic effect activity. The primary site of modification at this pH was His34. We developed a quantitative assay using sensitivity to neutral hydroxylamine to measure the proportion of urethane bonds involving carboxyalkylated histidines. This assay showed that histidine was pegylated preferentially at low pH levels with another protein, rh-Interleukin-10. CONCLUSIONS: Reaction pH can be used to select the preferred pegylation site chemistry.

Mass spectrometric mapping of disulfide bonds in recombinant human interleukin-13.

Interleukin 13 (IL-13), a member of the a-helical family of cytokines, has approximately 30% primary sequence homology with IL-4 and shares a common receptor component. The biologically active rhIL-13 is monomeric and non-glycosylated, and contains two disulfide bonds as determined by comparative electrospray mass spectrometric (MS) analysis of the protein before and after reduction with dithiothreitol-dithioerythritol. A trypsin-resistant core peptide of rhIL-13 was isolated and analyzed by plasma desorption (PD) MS, identifying a disulfide-linked core peptide. Subsequent digestion of this core peptide by pepsin, followed by PDMS analysis of the resulting cystine-containing peptic fragments, provided rapid determination of the existing disulfide bonds between cysteine residues 28-56 and 44-70. This disulfide arrangement is similar to that observed for the analogous four internal cysteine residues in hIL-4. The conservation of disulfide bond arrangements between hIL-13 and hIL-4, coupled with their alpha-helical structure and sequence homologies, confirms that IL-13 and IL-4 are structural homologues. It is also consistent with their reported similarities in biological function and receptor binding kinetics.

Expression, purification, and characterization of recombinant human interleukin-13 from NS-O cells.

Interleukin-13 is a cytokine which is secreted by activated T lymphocytes and primarily impacts monocytes, macrophages, and B cells. A synthetic gene coding for human interleukin-13 has been prepared and cloned into expression vector pEE12. The construct was transfected into NS-O cells, which showed stable expression of the recombinant protein. A four-step purification procedure consisting of S-Sepharose, Q-Sepharose, hydroxyapatite, and Sephacryl-100 chromatographies yielded bioactive interleukin-13 of > 98% purity. The purified protein was structurally characterized. The extinction coefficient at 280 nm was determined to be 5678 M-1 cm-1. Amino acid sequencing confirmed that the N-terminus of the purified protein was intact. Electrospray mass spectrometric analysis, size-exclusion chromatography, and SDS-PAGE revealed that the biologically active protein is monomeric and unglycosylated. Mass spectrometry and a chemical assay for free sulfhydryls indicated that the four cysteine residues of interleukin-13 are involved in two intramolecular disulfide bonds. The circular dichroism spectrum confirms that interleukin-13 belongs to the alpha-helical family of cytokines. A biologically inactive covalent trimer also forms in the cell culture, but can be separated from the monomer by the hydroxyapatite and size-exclusion chromatographies. These data indicate that human interleukin-13 retains many structural similarities to human interleukin-4, from which it arose by a gene duplication event.

Purification and characterization of 5-hydroxymethyluracil-DNA glycosylase from calf thymus. Its possible role in the maintenance of methylated cytosine residues.

5-Hydroxymethyluracil (HmUra) residues formed by the oxidation of thymine are removed from DNA through the action of a DNA glycosylase activity. This activity was purified over 1870-fold from calf thymus and found to be distinct from uracil (Ura)-DNA glycosylase. The HmUra-DNA glycosylase has a molecular weight of 38,000, a pH optimum of 6.7-6.8 and an apparent Km of 0.73 +/- 0.04 microM. These values are similar to those reported for other mammalian DNA glycosylases. The enzyme removed HmUra residues from single- and double-stranded DNA with almost equal efficiency. HmUra-DNA glycosylase activity was not product inhibited by free HmUra. The DNA glycosylase activity was inhibited by Mg2+, but the purest enzyme fractions contained a Mg2+-dependent apurinic/apyrimidinic endonuclease activity. HmUra-DNA glycosylase and the recently described 5-hydroxymethylcytosine (HmCyt)-DNA glycosylase (Cannon, S. V., Cummings, A. C., and Teebor, G. W. (1988) Biochem. Biophys. Res. Commun. 151, 1173-1179) are unique among known DNA glycosylases in being present in mammalian cells and absent from bacteria. These DNA glycosylase activities were shown here to reside on different proteins. We suggest that the major function of HmUra-DNA glycosylase, together with HmCyt-DNA glycosylase, is the maintenance of methylated cytosine residues in the DNA of higher organisms.