The identification of free cysteine residues within antibodies and a potential role for free cysteine residues in covalent aggregation because of agitation stress.

Human immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) antibodies contain multiple disulfide bonds, which are an integral part of the structure and stability of the protein. Open disulfide bonds have been detected in a number of therapeutic and serum derived antibodies. This report details a method that fluorescently labels free cysteine residues, quantifies, and identifies the proteolytic fragments by liquid chromatography coupled to online mass spectrometry. The majority of the open disulfide bonds in recombinant and serum derived IgG1 and IgG2 antibodies were in the constant domains. This method was applied to the identification of cysteines in an IgG2 antibody that are involved in the formation of covalent intermolecular bonds because of the application of a severe agitation stress. The free cysteine in the CH 1 domain of the IgG2 decreased upon application of the stress and implicates open disulfide bonds in this domain as the likely source of free cysteines involved in the formation of intermolecular disulfide bonds. The presence of comparable levels of open disulfide bonds in recombinant and endogenous antibodies suggests that open disulfide bonds are an inherent feature of antibodies and that the susceptibility of intermolecular disulfide bond formation is similar for recombinant and serum-derived IgG antibodies.

Co-expression as a convenient method for the production and purification of core histones in bacteria.

Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)(2) heterotetrameric complex if they are co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2) can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co-expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity.

Depression of Saccharomyces cerevisiae invasive growth on non-glucose carbon sources requires the Snf1 kinase.

Haploid Saccharomyces cerevisiae cells growing on media lacking glucose but containing high concentrations of carbon sources such as fructose, galactose, raffinose, and ethanol exhibit enhanced agar invasion. These carbon sources also promote diploid filamentous growth in response to nitrogen starvation. The enhanced invasive and filamentous growth phenotypes are suppressed by the addition of glucose to the media and require the Snf1 kinase. Mutations in the PGI1 and GND1 genes encoding carbon source utilization enzymes confer enhanced invasive growth that is unaffected by glucose but requires active Snf1. Carbon source does not modulate FLO11 flocculin expression, but enhanced polarized bud site selection is necessary for invasion on certain carbon sources. Interestingly, deletion of SNF1 blocks invasion without affecting bud site selection. Snf1 is also required for formation of spokes and hubs in multicellular mats. To examine glucose repression of invasive growth more broadly, we performed genome-wide microarray expression analysis in wild-type cells growing on glucose and galactose, and snf1 Delta cells on galactose. SNF1 probably mediates glucose repression of multiple genes potentially involved in invasive and filamentous growth. FLO11-independent cell-cell attachment, cell wall integrity, and/or polarized growth are affected by carbon source metabolism. In addition, derepression of cell cycle genes and signalling via the cAMP-PKA pathway appears to depend upon SNF1 activity during growth on galactose.

Peptide chips for the quantitative evaluation of protein kinase activity.

Peptide chips are an emerging technology that could replace many of the bioanalytical methods currently used in drug discovery, diagnostics, and cell biology. Despite the promise of these chips, their development for quantitative assays has been limited by several factors, including a lack of well-defined surface chemistries to immobilize peptides, the heterogeneous presentation of immobilized ligands, and nonspecific adsorption of protein to the substrate. This paper describes a peptide chip that overcomes these limitations, and demonstrates its utility in activity assays of the nonreceptor tyrosine kinase c-Src. The chip was prepared by the Diels-Alder-mediated immobilization of the kinase substrate AcIYGEFKKKC-NH(2) on a self-assembled monolayer of alkanethiolates on gold. Phosphorylation of the immobilized peptides was characterized by surface plasmon resonance, fluorescence, and phosphorimaging. Three inhibitors of the enzyme were quantitatively evaluated in an array format on a single, homogeneous substrate.