Michiko Kato

Systems for the detection and analysis of protein–protein interactions

The analysis of protein–protein interactions is important for developing a better understanding of the functional annotations of proteins that are involved in various biochemical reactions in vivo. The discovery that a protein with an unknown function binds to a protein with a known function could provide a significant clue to the cellular pathway concerning the unknown protein. Therefore, information on protein–protein interactions obtained by the comprehensive analysis of all gene products is available for the construction of interactive networks consisting of individual protein–protein interactions, which, in turn, permit elaborate biological phenomena to be understood. Systems for detecting protein–protein interactions in vitro and in vivo have been developed, and have been modified to compensate for limitations. Using these novel approaches, comprehensive and reliable information on protein–protein interactions can be determined. Systems that permit this to be achieved are described in this review.

Construction of a novel synergistic system for production and recovery of secreted recombinant proteins by the cell surface engineering

We determined whether the cocultivation of yeast cells displaying a ZZ-domain and secreting an Fc fusion protein can be a novel tool for the recovery of secreted recombinant proteins. The ZZ-domain from Staphylococcus aureus protein A was displayed on the cell surface of Saccharomyces cerevisiae under the control of the GAL1 promoter. Strain S. cerevisiae BY4742 cells displaying the ZZ-domain on their surface were used for cocultivation with cells that produce a target protein fused to the Fc fragment as an affinity tag. The enhanced green fluorescent protein or Rhizopus oryzae lipase was genetically fused to the N and C termini of the Fc fragment of human immunoglobulin G, respectively. Through analysis by fluorescence-activated cell sorting and enzymatic assay, it was demonstrated that these fusion proteins are successfully produced in the medium and recovered by affinity binding with the cell surface displaying the ZZ-domain. These results suggest that the ZZ-domain-displaying cell and Fc fusion protein-secreting cell can be applied to use in synergistic process of production and recovery of secreted recombinant proteins.

Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering

To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EG II) from Trichoderma reesei, a combinatorial library of the cellulose‐binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid‐swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5‐fold) had higher hydrolysis activity (1.3‐fold) compared to the parent EG II‐displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.

Analysis of a processing system for proteases using yeast cell surface engineering: conversion of precursor of proteinase A to active proteinase A

The display of a protease, carboxypeptidase Y (CPY) or procarboxypeptidase Y (proCPY), which is the vacuolar protease, on the yeast-cell surface was successfully performed using yeast-cell-surface engineering for the first time. Through that we could confirm the processing of vacuolar proteases containing proteinase A (PrA) and proteinase B (PrB) which are related to the maturation of proCPY, using a novel cell-surface engineering technique. Various protease-knockout strains of Saccharomyces cerevisiae with the CPY-displaying system were constructed to evaluate the operation of the activation process of CPY. The display of CPY (CPY-agg, which is a fusion protein of CPY with C-terminal half of α-agglutinin) on the cell surface was confirmed by immunofluorescence staining. The activity of the CPY-agg was determined after the conversion of proCPY to active CPY by treatment of whole cells with proteinase K. In the proCPY-displaying CPY-knockout strain and PrB-knockout strain, CPY was displayed as an active (mature) form, but in the proCPY-displaying PrA-knockout strain, CPY was present as an inactive form (proCPY). These facts indicate that PrA had been already activated before its transport to the vacuole and that active mature PrA might convert proCPY to CPY before the transport of proCPY to the vacuole. From these results, it was suggested that by using the yeast-cell-surface engineering at the location of the initial step, the autocatalytic activation from proPrA to PrA might occur before the vacuolar branch separates from the main secretory pathway.

Construction of a Cultivation System of a Yeast Single Cell in a Cell Chip Microchamber

A novel single cell screening system was constructed using a yeast cell chip in combination with the yeast cell surface engineering [NanoBiotechnology 2005, 1 , 105–111]. Enzymes or functional proteins displayed on a yeast cell surface can be used as a protein cluster. To achieve high‐throughput screening of protein libraries on the cell surface, a catalytic reaction by a single cell‐surface‐engineered yeast cell was successfully carried out in the microchamber on the yeast cell chip. After screening, to replicate a target cell for use in measuring of activity, DNA sequencing, and preservation, a novel single cell cultivation system in the yeast cell chip was constructed. To avoid damage of the rapid dry up of medium in the microchamber array, the yeast cell chip was modified with a protection sheet, so that the modified chip was like a micro‐culture tank constructed on the yeast cell chip microchamber. As a result, single yeast cell cultivation in the yeast cell chip microchamber was observed, and the modified yeast cell chip was evaluated to be good for a single cell selection. The improvement showed that the single cell screening system coupled with the single cell cultivation using the modified yeast cell chip may be superior to that by a cell sorter for the isolation of a target cell and its practical use.

Effects of high pressure and temperature on micelle formation of sodium deoxycholate and sodium dodecylsulfate

tions and changes the fluidity of membranes. For example, membrane intrinsic protein (Na, K)-ATPase is inhibited by increasing hydrostatic pressure, because increased pressure decreases the membrane fluidity to inhibit the conformational transition necessary for the (Na, K)-ATPase reaction (1). However, the effect of pressure on the lipid component in cell membranes of living organisms is not well known. For full understanding of this subject, studies of the behavior of natural and artificial lipids under high pressure are required. In this study, sodium deoxycholate (DOC) and sodium dodecylsulfate (SDS) are used as a model of lipids, and critical micelle concentration (CMC) under high pressure is measured to investigate the pressure effects on the aggregation or the hydrophobic interaction of lipid molecules. Absorbance under high pressure was measured with a thermocontrolled high-pressure photometer cell with sapphire windows (Teramecs Co., Kyoto, Japan) which is connected with a hand-type high-pressure pump and attached to a ultraviolet (UV)-visible spectrophotometer (UV-2500PC; Shimadzu Co., Kyoto, Japan). A series of aqueous amphiphile solutions containing 48.4 μM Coomassie brilliant blue R-250 (Nacalai Tesque, Inc., Kyoto, Japan) were made according to the method of Courtney et al. (2), and 1.5 mL of the sample solution was introduced to the high-pressure cell and incubated for 3–10 min at a given temperature and pressure. Then, the absorbance at 596 nm was measured. It was checked for 15 min to obtain the stable value. The wavelength of maximum dye absorption shifts from 550 to 596 nm when amphiphiles form micelles. CMC was determined as the break point when the absorbance at 596 nm vs. the logarithm of amphiphile concentration was plotted on a graph. CMC values of various amphiphiles determined by the Coomassie brilliant blue method at 25°C are compared with those of other methods including the conductivity method in Table 1. The values obtained by the dye method are in good agreement with those reported in the literature, except for a notably different value from Reference 3. Furthermore, it was confirmed that change of the dye concentration (12.1–48.4 μM) caused no change in CMC values obtained. Thus, the dye method employed in the present experiments is considered to be reliable. The CMC of DOC and SDS at various pressures up to 400 MPa and at temperatures of 20 to 45°C were determined (Fig. 1). In case of SDS, the pressure dependency at various temperatures was in fairly good agreement with published data (3), although the dye method tended to give somewhat lower values than the conductivity method. At any temperature, pressure dependency showed a convex curve with maximum at 100 MPa as already reported with SDS (7). The CMC of DOC increased slightly up to 100 MPa and then decreased at 100 MPa or higher at 25, 35, and 45°C (Fig. 1). However, a strong decrease in the CMC was observed at 100 MPa or

Substrate specificity of rat brain neurolysin disclosed by molecular display system and putative substrates in rat tissues

To search for the substrates, other than neurotensin, of rat brain neurolysin, a novel method of determining peptidase activity was developed using a yeast molecular display system. This is a useful and convenient method of handling homogenously pure proteins to evaluate the properties of neurolysin. The neurolysin gene was ligated to the C-terminal half of the α-agglutinin gene with a FLAG tag sequence and a yeast cell-surface molecular displaying plasmid was constructed. Display of neurolysin with correct folding and appropriate activity was verified by immunofluorescence staining and activity measurement of a bradykinin-related peptide. The cleavage sites of peptides were determined by high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The results showed the amino acid preferences of hydrophobic, aromatic, and basic residues, which were the same as those of soluble neurolysin. Moreover, this method clearly showed the presence of two recognition motifs in neurolysin. By using these motifs, novel substrate candidates of neurolysin in rat tissues were screened, and several bioactive peptides that regulate feeding were found. We also discussed the ubiquitous distribution of neurolysin in rat tissues and the functions of substrate candidate peptides.

Construction of a selective cleavage system for a protein displayed on the cell surface of yeast

We constructed a novel protein-purification system in which Saccharomyces cerevisiae with a protein displayed on the cell surface is harvested and the displayed protein is then cleaved from the cell surface. GFPuv was used as a model protein in this cell surface engineering experiment. In this system, the C-terminal 320 amino acids of α-agglutinin were bound to the C-terminal of GFPuv for display on the cell surface. In this novel system, the insertion of the recognition sequence-encoding gene of protease factor Xa between GFPuv and α-agglutinin was successfully carried out. The GFPuv, displayed by the insertion, was successfully cleaved from yeast cell surface by treatment with factor Xa, and could be easily recovered. By removing such a protease with well-known properties, the displayed protein could be isolated and purified with relative ease.

Metallopeptidase, neurolysin, as a novel molecular tool for analysis of properties of cancer-producing matrix metalloproteinases-2 and -9.

To compare the substrate preferences of rat brain neurolysin and cancer-producing matrix metalloproteinases (MMPs), which have the same architecture in their catalytic domains, the cleavage activity of neurolysin toward MMP-specific fluorescence-quenching peptides was quantitatively measured. The results show that neurolysin effectively cleaved MOCAc [(7-methoxy coumarin-4-yl) acetyl]-RPKPYANvaWMK(Dnp[2,4-dinitrophenyl])-NH2, a specific substrate of MMP-2 and MMP-9, but hardly cleaved MOCAc-RPKPVENvaWRK(Dnp)-NH2, a specific substrate of MMP-3, suggesting that neurolysin has a similar substrate preference to MMP-2 and MMP-9. A structural comparison between neurolysin and MMP-9 showed the similar key amino acid residues for substrate recognition. The possible application of neurolysin displayed on the yeast cell surface, as a safe protein alternative to MMP-2 and MMP-9 which induce cancer cell growth, invasion, and metastasis, to analysis of properties of the MMPs, including the screening of inhibitors and analysis of inhibition mechanism etc., are also discussed.

Construction of novel single-cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries

A novel screening system using a microchamber array chip was developed for construction of combinatorial nano-sized protein libraries in combination with yeast cell surface engineering. It is possible to place a single yeast cell into each microchamber, to observe its behavior, and to pick up the target cell. The microchamber array chip is referred to as a “yeast cell chip.” A single EGFP-displaying yeast cell could be detected, picked up by a micro-manipulator, and cultivated on agar medium. Furthermore, a catalytic reaction, the hydrolysis of fluorescein dioctanate, by a single yeast cell displaying Rhizopus oryzae lipase (ROL) was carried out in one microchamber. The ROL-encoding gene in a single ROL-displaying cell was amplified by PCR. These results demonstrate that this yeast cell chip in combination with cell surface engineering could be used as a tool in a high-throughput screening system not only for a single living cell and a whole-cell catalyst with a nano-sized protein cluster but also for modified nano-sized and functional protein molecules from protein libraries on the cell surface.