Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04626 (erbB-2)
 5,251 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations.
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PMID:Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis. 1590 61

A new method that allows fast and quantitative recovery of hydrophobic or amphipathic peptides, or both, after their intimate incorporation into lipid membranes, is proposed. It relies on the use of small Sep-Pak cartridges and simple chromatographic handling. Peptides selected for this study are the 35 amino acid transmembrane domain of the Neu/erbB-2 protein and its point mutated (V664E) analogue expressed in some cancers, the 25 amino acid BH4 domain from the Bcl-2 antiapoptotic protein and the 15 amino acid Catestatin segment from chromogranin A found to have antimicrobial capabilities. Incorporation of peptides into membranes is accomplished using organic solvent cosolubilization and several cycles of freeze-drying/hydration from aqueous solution. For the hydrophobic peptides, separation from the membrane is performed on Sep-Pak C2 columns in two steps: (i) water/methanol elution of lipids and (ii) peptide elution using aprotic solvents (acetonitrile, 2-propanol). For amphipathic peptides, separation is performed on Sep-Pak C(18) columns using selective elution in one single step: water/methanol elution to recover first the peptide and then the lipids. Peptide and lipid recovery after all purification steps range from 60 to 80%, with peptide purity above 96%. This new method is simple, inexpensive, and very fast: a 10-mg membranous mixture containing 10% (w/w) peptide may be separated in 20-30 min.
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PMID:Fast and quantitative recovery of hydrophobic and amphipathic peptides after incorporation into phospholipid membranes. 1687 68