Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.     

Processing and maturation of carboxypeptidase Y and alkaline phosphatase in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins.     

Molecular cloning and structural characterization of the hagfish proteinase inhibitor of the alpha-2-macroglobulin family

The most primitive living vertebrate the hagfish has a dimeric proteinase inhibitor, a protein homologous to human ₂-macroglobulin, in its plasma at high concentration. Although the hagfish proteinase inhibitor has been isolated and its function and quaternary structure studied, its primary structure, subunit composition and fragmentation process remain unclear. In this study, hagfish proteinase inhibitor cDNA was cloned, sequenced and cDNA-deduced amino acid sequence was analyzed. A large fraction of homosubunits in the dimeric structure of the protein has undergone a cleavage at a specific arginyl residue (Arg⁸³³) while the rest retained their chain integrity without being processed. Thus random combinations of processed and nonprocessed subunits in the dimeric structure of this protein result in different molecular conformers and generate a complicated multiband pattern in SDS-PAGE. It was further demonstrated by proteolytic analysis that the hagfish inhibitor has no susceptible arginyl residues within its bait region and thus incapable of trapping arginine specific proteinases. This implies that the specific subunit cleavage at Arg⁸³³ was caused by an unknown arginine specific proteinase which escaped from the entrapment by the hagfish inhibitor.     

Nanotechnology and Protein Mechanics

The atomic force microscope is currently used in our and many other laboratories to measure the mechanical response of polypeptide and proteins against tensile forces applied to well defined positions in their chemical structures. The resulting force vs. extension (F-E) curves are analyzed in relation to their known conformations under various conditions. The method can be extended to study the mechanical responses of other, often much larger biological structures, and extract the component proteins and DNAs from cell membranes and chromosomes.     

蝙蝠在维护生态系统中的作用

蝙蝠是世界上分布最广、种群数量最多、进化最成功的哺乳动物类群之一,它们在维护生态系统中具有重要的作用：蝙蝠在生态系统中占据了独特的生存空间;蝙蝠是许多农、林及卫生害虫的天敌;蝙蝠是种子的传播者和花粉的传授者;在一定生态系统中蝙蝠可能起到关键种的作用;食虫蝙蝠粪便和酮体均是我国传统中药中的主要药材之一。研究和保护蝙蝠在维护生态环境中具有十分重要的意义。     

Force measurement for antigen-antibody interaction by atomic force microscopy using a photograft-polymer spacer

To determine the intermolecular force on protein-protein interaction (PPI) by atomic force microscopy (AFM), a photograft-polymer spacer for protein molecules on both surfaces of the substrate and AFM probe tip was developed, and its effectiveness was assessed in a PPI model of a pair of human serum albumin (HSA) and its monoclonal antibody (anti-HSA). A carboxylated photoiniferter, N-(dithiocarboxy)sarcosine, was derivatized on both surfaces of the glass substrate and AFM probe tip, and subsequently water-soluble nonionic vinyl monomers, N,N-dimethylacrylamide (DMAAm), were graft-polymerized on them upon ultraviolet light irradiation. DMAAm-photograft-polymerized spacers with carboxyl groups at the growing chain end but with different chain lengths on both surfaces were prepared. The proteins were covalently bound to the carboxyl terminus of the photograft-polymer chain using a water-soluble condensation agent. The effects of the graft-spacer length on the profile of the force-distance curves and on the unbinding characteristics (unbinding force and unbinding distance) were examined in comparison with those in the case of the commercially available poly(ethylene glycol) (PEG) spacer. The frequency of the nonspecific adhesion force profile was markedly decreased with the use of the photograft spacers. Among the force curves detected, a high frequency of single-peak curves indicating the unbinding process of a single pair of proteins and a very low frequency of multiple-peak profiles were observed for the photograft spacers, regardless of the graft chain length, whereas a high frequency of no-force peaks was noted. These observations were in marked contrast with those for the PEG spacer. The force peak values determined ranged from 88 to 94 pN, irrespective of the type of spacer, while the standard deviation of force distribution observed for the photograft spacer was lower than that for the PEG spacer, indicating that the photograft spacers provide a higher accuracy of force determination.     

Engineering of protein secretion in yeast: strategies and impact on protein production

Yeasts combine the ease of genetic manipulation and fermentation of a microorganism with the capability to secrete and modify foreign proteins according to a general eukaryotic scheme. Their rapid growth, microbiological safety, and high-density fermentation in simplified medium have a high impact particularly in the large-scale industrial production of foreign proteins, where secretory expression is important for simplifying the downstream protein purification process. However, secretory expression of heterologous proteins in yeast is often subject to several bottlenecks that limit yield. Thus, many studies on yeast secretion systems have focused on the engineering of the fermentation process, vector systems, and host strains. Recently, strain engineering by genetic modification has been the most useful and effective method for overcoming the drawbacks in yeast secretion pathways. Such an approach is now being promoted strongly by current post-genomic technology and system biology tools. However, engineering of the yeast secretion system is complicated by the involvement of many cross-reacting factors. Tight interdependence of each of these factors makes genetic modification difficult. This indicates the necessity of developing a novel systematic modification strategy for genetic engineering of the yeast secretion system. This mini-review focuses on recent strategies and their advantages for systematic engineering of yeast strains for effective protein secretion.     

Spring mechanics of alpha-helical polypeptide

To design protein- and polymer-based micro-machineries, it is important to understand the mechanical properties of basic structural elements such as the alpha-helix of polypeptides. We employed the force measurement mode of an atomic force microscope (AFM) to investigate the spring mechanics of poly-L-glutamic acid (PGA) in its helical and randomly coiled states. After covalently anchoring the polypeptide between a silicon substrate and an AFM tip, the force required to stretch the polymer was measured. The results indicated that PGA in its helical conformation could be stretched almost fully with a continuous increase in the stretching force, suggesting that it can be used as a reliable coil-spring in the future design of spring-loaded molecular machineries.     

Enhanced productivity of protease-sensitive heterologous proteins by disruption of multiple protease genes in the fission yeast Schizosaccharomyces pombe

The creation of protease-deficient mutants to avoid product degradation is one of the current strategies employed to improve productivity and secretion efficiency of heterologous protein expression. We previously constructed a set of single protease-deficient mutants of the fission yeast Schizosaccharomyces pombe by respective disruption of 52 protease genes, and we succeeded in confirming useful disruptants (Idiris et al., Yeast 23:83–99, 2006). In the present study, we attempted multiple deletions of 13 protease genes, single deletions of which were previously confirmed as being beneficial for reducing extracellular product degradation. Using PCR-based gene replacement, a series of multiple deletion strains was constructed by multiple disruption of a maximum of seven protease genes. Effects of the resultant multiple deletion strains on heterologous expression were then measured by practical expression of a proteolytically sensitive model protein, the human growth hormone (hGH). Time profiles of hGH secretion from each resultant mutant demonstrated significantly enhanced hGH productivity with processing of the multiple protease deletions. The data clearly indicated that disruption of multiple protease genes in the fission yeast is an effective method for controlling proteolytic degradation of heterologous proteins particularly susceptible to proteases.     

Luminescence and structural studies of yttrium and heavier lanthanide-picrate complexes with pentaethylene glycol

The monoclinic structural isomers with molecular formula of [M(Pic)₂(EO5)](Pic) where EO5 = pentaethylene glycol, Pic = picrate anion, and M = Gd, Tb, Er, Tm, Yb, and Y have been synthesized and characterized. The current study was conducted in the solid state to evaluate the coordination pattern of the central metal ion with the EO5 ligand in the presence of Pic anion. The photoluminescence (PL) spectra of the Tb and Yb complexes have the typical 4f-4f transition emissions of Tb(III) and Yb(III) ions. The Gd, Er, Tm, and Y complexes had broad bands resulting from the ligands. The counteranion factor influenced the emission intensity in the Tb-Pic-EO5 and Tb-NO₃-EO5 complexes in several solvents also were studied. The Pic anion acts as a quencher in the [Tb(Pic)₂(EO5)](Pic) complex due to the nitro withdrawing groups was clearly observed both in solution and the solid state.