Michael Manneberg

Effect of the hydrolysis method on the determination of the amino acid composition of proteins

Fast and reproducible separation and determination of amino acids serves the economical and reliable characterization and quantification of peptides and proteins as well as the identification of proteins by amino acid composition analysis on a large-scale. A prerequisite of a successful compositional analysis is a complete hydrolysis of the peptides and proteins and a quantitative recovery of the residues in the hydrolyzate. We investigated the effect of different acid-hydrolysis methods on the compositional analysis of known proteins in solution and after blotting onto polyvinylidene difluoride membranes and worked out the conditions for the processing of large numbers of samples. The reliability of each method was studied by introducing the analysis data into the AACompIdent software and deducing the protein identification scores. All acid-hydrolysis methods delivered reliable analysis data. The most accurate data were provided by conventional, thermal hydrolysis of proteins in solution in the presence of methanesulfonic acid, closely followed by hydrolysis with hydrochloric acid and microwave radiation-dependent hydrolysis with hydrochloric or methanesulfonic acid, respectively. For blotted proteins, conventional hydrolysis delivered more accurate analysis data in comparison with the microwave radiation-induced hydrolysis. The extraction of the residues from the membrane hydrolyzate was a critical step for unambiguous protein identification. Microwave radiation-induced hydrolysis was responsible for a higher degree of racemization of the residues.

Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins

The concentrations of several non-glycosylated and glycosylated recombinant and native proteins were determined by three widely used colorimetric methods: Coomassie brilliant blue, bicinchoninic acid and Lowry, and, for comparison, by amino acid composition analysis. The colorimetric methods gave results differing from the values derived from the amino acid analysis, in some cases by up to 60%. For the non-glycosylated recombinant proteins, the results were in relatively good agreement with each other and with the values determined on the basis of the amino acid analysis. The Coomassie blue method was strongly dependent on the hydrophobicity of the individual protein. The bicinchoninic acid method gave results closest to those of the amino acid analysis. For the glycosylated proteins, both recombinant and native, the Coomassie blue assay gave values lower, whereas the two other methods gave values higher than those determined on the basis of the amino acid analysis. The concentration of a recombinant interferon gamma receptor produced in two differently glycosylated forms was underestimated by the Coomassie blue assay and overestimated by the bicinchoninic acid and Lowry methods, while for the non-glycosylated form of the same protein, the three colorimetric methods delivered comparable values. The results suggest a potential interference of protein glycosylation with the colorimetric assays.

Identification of a human immunodeficiency virus-1 protease cleavage site within the 66,000 dalton subunit of reverse transcriptase

The human immunodeficiency virus-1 reverse transcriptase is a heterodimer of related 51 and 66 kDa subunits. The smaller subunit arises by viral protease-catalyzed cleavage of the carboxy-terminal domain of the 66 kDa species. Comparison of the amino acid composition analyses of the isolated 51 kDa and 66 kDa subunits indicates that the carboxyl terminus of 51 kDa is Phe440. This site was confirmed in vitro using purified recombinant protease and a peptide spanning the postulated cleavage area. The sequence surrounding this site does not show significant homology to other protease cleavage sites in the viral gag and pol precursors; thus, this new information may contribute to our understanding of the sequence specificity of the viral protease.

Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells. Purification, biochemical characterization, and stoichiometry of binding.

Agents that antagonize the functions of interferon γ (IFNγ) are potential pharmaceuticals against several immunological and inflammatory disorders. IFNγ receptor-immunoglobulin G fusion proteins (IFNγR-IgG) function as antagonists of endogenous IFNγ and have longer half-lives in vivo in comparison with soluble IFNγ receptors (sIFNγR), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFNγ receptor and the hinge, CH2 and CH3 domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFNγR-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFNγR-IgG3 bound IFNγ in solid phase assays and ligand blots, competed for the binding of radiolabeled IFNγ to the cell surface receptor of Raji cells, and inhibited the IFNγ-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFNγR-IgG3 fusion proteins bound two IFNγ dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFNγR-IgG3 fusion protein did not precipitate IFNγ. Dissociation of bound IFNγ from IFNγR-IgG3 was 2-fold slower than from the sIFNγR produced in insect cells.