Kinetics of Antibody Aggregation at Neutral pH and Ambient Temperatures Triggered by Temporal Exposure to Acid

The purification process of an antibody in manufacturing involves temporal exposure of the molecules to low pH followed by neutralizationpH-shift stresswhich causes aggregation. It remains unclear how aggregation triggered by pH-shift stress grows at neutral pH and how it depends on the temperature in an ambient range. We used static and dynamic light scattering to monitor the time-dependent evolution of the aggregate size of the pH-shift stressed antibody between 4.0 and 40.0 °C. A power-law relationship between the effective molecular weight and the effective hydrodynamic radius was found, indicating that the aggregates were fractal with a dimension of 1.98. We found that the aggregation kinetics in the lower-temperature range, 4.0–25.0 °C, were well described by the Smoluchowski aggregation equation. The temperature dependence of the effective aggregation rate constant gave 13 ± 1 kcal/mol of endothermic activation energy. Temporal acid exposure creates an enriched population of unfolded protein molecules that are competent of aggregating. Therefore, the energetically unfavorable unfolding step is not required and the aggregation proceeds faster. These findings provide a basis for predicting the growth of aggregates during storage under practical, ambient conditions

Artificial Mating-Type Conversion and Repetitive Mating for Polyploid Generation

The yeast <i>Saccharomyces cerevisiae</i> is one of the best-understood biological systems and can produce numerous useful compounds. Sexual hybridization (mating) can drive dramatic evolution of yeasts by the inheritance of half of the parental genomic information from each cell. Unfortunately, half of the parental genomic information is lost in individual cells in the next generation. Additionally, recombination of homologous chromosomes during meiosis gives rise to diversity in the next generation; hence, it is commonly employed to identify targets from diverse cell populations, based on the mating machinery. Here, we established a system for generating polyploids that inherit all genetic information from the parental strains <i>via</i> artificial mating-type conversion and repetitive mating. We prepared α-type haploid strains whose chromosomes were tagged with genes encoding fluorescent proteins or transcriptional factors. Only the mating-type locus was successfully converted from α-type to <b>a</b>-type sequence by the endonuclease Ho, and the resultant <b>a</b>-type cells mated with each α-type haploid to yield an <b>a</b>/α-type diploid strain with all genetic information from both parental strains. Importantly, we repeatedly converted the mating-type of polyploid cells to obtain <b>a</b>-type cells capable of mating with α-type cells. This approach can potentially facilitate yeast-strain development with unparalleled versatility, utilizing vast available resources

Rapid Evaluation of Tyrosine Kinase Activity of Membrane-Integrated Human Epidermal Growth Factor Receptor Using the Yeast Gγ Recruitment System

Epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family and plays key roles in the regulation of fundamental cellular processes, including cell proliferation, migration, differentiation, and survival. Deregulation of EGFR tyrosine kinase activity is involved in the development and progression of human cancers. In the present study, we attempted to develop a method to evaluate the tyrosine kinase activity of human EGFR using the yeast Gγ recruitment system. Autophosphorylation of tyrosine residues on the cytoplasmic tail of EGFR induces recruitment of Grb2-fused Gγ subunits to the inner leaflet of the plasma membrane in yeast cells, which leads to G-protein signal transduction and activation of downstream signaling events, including mating and diploid cell growth. We demonstrate that our system is applicable for the evaluation of tyrosine kinase inhibitors, which are regarded as promising drug candidates to prevent the growth of tumor cells. This approach provides a rapid and easy-to-use tool to select EGFR-targeting tyrosine kinase inhibitors that are able to permeate eukaryotic membranes and function in intracellular environments

Imparting Albumin-Binding Affinity to a Human Protein by Mimicking the Contact Surface of a Bacterial Binding Protein

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (<i>K</i>_D = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13–15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another

Frequency of each amino acid residue at positions 7 and 8 before and after diploid growth screening.

<p>Pi indicates initial population, Pf indicates final population, Er indicates Enrichment ratio, and symbol * indicates amber stop codon.</p

Contribution of N-terminal positions 7 to 10 to the efficiency of dual lipidation.

<p>The diploid growth assay was used to test the mating ability of (A) yeast cells expressing MP6-Gγ_cyto, MP6L-Gγ_cyto, MP10Y-Gγ_cyto, MP10Hii-Gγ_cyto or MP10Hiii-Gγ_cyto, and (B) those expressing MP8YL-Gγ_cyto or MP6L2Y-Gγ_cyto.</p

Reliability of predicted amino acid residue preferences.

<p>The membrane-targeting ability of each Gγ_cyto-hybrid was quantitatively measured using the diploid growth assay. Standard deviations of three independent experiments are presented. DW-Gγ_cyto (lane 1), GE-Gγ_cyto (lane 2), WA-Gγ_cyto (lane 3), MP10Y-Gγ_cyto (lane 4), TM-Gγ_cyto (lane 5), QT-Gγ_cyto (lane 6), FN-Gγ_cyto (lane 7).</p

Artificial Conversion of the Mating-Type of <i>Saccharomyces cerevisiae</i> without Autopolyploidization

Crossbreeding is a classical yeast hybridization procedure, where the mating of haploid cells of opposite mating-type, <i>MAT</i>a and <i>MATα</i> cells, produces a new heterozygous diploid. Here, we describe a method to generate haploid <i>MAT</i>a and <i>MATα</i> cells using mating-type conversion caused by expression of the <i>HO</i> gene, which encodes an endonuclease. Importantly, our method prevents the autopolyploidization that typically arises during artificial mating-type conversion. This facilitates isolation of the desired mating-type of yeast cells with simple and easy procedure. In the current study, we designed a suitable genetic circuit for each haploid cell and converted <i>MAT</i>α haploid cells into <i>MAT</i>a haploid cells and vice versa, demonstrating the utility of constructed artificial regulation network to prevent autopolyploidization. Via forced expression of the a1 gene in <i>MAT</i>α haploid cells or of α2 in <i>MAT</i>a haploid cells, the undesirable mating ability of yeast cells was completely suppressed. We confirmed the success in prevention of autopolyploidization by ploidy analysis. This new approach provides a reliable and versatile tool for yeast crossbreeding, so that it will be useful for scientific research and industrial applications of yeast

New approach to investigate membrane associations of proteins utilizing the yeast G-protein signal transduction.

<p>Wild-type Gγ is lipid-modified at its C-terminus, and localized at plasma membrane to transmit the intracellular signal. An engineered Gγ lacking membrane association (Gγ_cyto) is fused to the target protein domain or peptide motif, yielding a Gγ_cyto hybrid protein. When the target protein domain or peptide motif does not confer membrane association, G-protein signaling is not restored. In contrast, when a Gγ_cyto hybrid protein confers plasma membrane localization, G-protein signaling is restored, leading to induction of the mating response and generation of diploid cells.</p

Amino acid residue preferences at positions 7 and 8 for N-terminal dual lipidation.

<p>Frequency of each amino acid residue at (A) position 7 and (B) position 8 in plasmids extracted from 100 randomly picked colonies. The symbol * indicates amber stop codon. Black columns, frequencies before screening (initial); gray columns, frequencies after screening (final).</p