Genetic immobilization of cellulase on the cell surface of Saccharomyces cerevisiae

We tried genetically to immobilize cellulase protein on the cell surface of the yeast Saccharomyces cerevisiae in its active form. A cDNA encoding FI-carboxymethylcellulase (CMCase) of the fungus Aspergillus aculeatus, with its secretion signal peptide, was fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast α-agglutinin, a protein involved in mating and covalently anchored to the cell wall. The plasmid constructed containing this fusion gene was introduced into S. cerevisiae and expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase activity was detected in the cell pellet fraction. The CMCase protein was solubilized from the cell wall fraction by glucanase treatment but not by sodium dodecyl sulphate treatment, indicating the covalent binding of the fusion protein to the cell wall. The appearance of the fused protein on the cell surface was further confirmed by immunofluorescence microscopy and immunoelectron microscopy. These results proved that the CMCase was anchored on the cell wall in its active form. Received: 19 March 1997 / Received revision: 19 May 1997 / Accepted: 1 June 1997     

Lactic acid bacteria display on the cell surface cytosolic proteins that recognize yeast mannan

Fluorescent-labeled invertase, a hyperglycosylated mannoprotein from Saccharomyces cerevisiae, was found to bind to Lactococcus lactis IL1403 at acidic pH. Proteins on the cell wall of the bacterium affinity-purified using invertase as a ligand were identified to be heat shock proteins such as DnaK and GroEL and glycolytic enzymes such as pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase. DnaK bound to both the bacterium and yeast at pH 4 and aggregated them at above 0.1 mg/ml, whereas no significant difference between the circular dichroism spectra of DnaK at pH 4 and 7 was observed. These results indicate that the cytosolic proteins, including DnaK displayed on the cell wall, cause the lactic acid bacterium to adhere to the yeast.     

Studies of the cell surface of Paramecium. Ciliary membrane proteins and immobilization antigens.

We have developed a procedure to isolate the ciliary membranes of Paramecium and have analysed the membrane proteins by electrophoresis on polyacrylamide gels containing either Triton X-100 or sodium dodecyl sulphate. The electrophoretic pattern on gels containing sodium dodecyl sulphate showed 12-15 minor bands of mol.wt. 25 000-150 000 and on major band of mol.wt. 200 000-300 000 that contained approximately three-quarters of the total membrane protein. 2. We present evidence that the major membrane protein is related to, but not identical with, the immobilization antigen (i-antigen), which is a large (250 000 mol.w.), soluble, surface protein of Paramecium. The similarity of the i-antigen and the major membrane protein was shown by immunodiffusion and by the electrophoretic mobilities in sodium dodecyl sulphate of these two proteins from Paramecium of serotypes A and B. The non-identity of these two proteins was shown by their different electrophoretic mobilities on Triton X-100 containing gels and their different solubilities. 3. We propose that the major membrane protein and the i-antigen have a precursor-product relationship.     

Genetic and proteomic evidences support the localization of yeast enolase in the cell surface

Although enolase, other glycolytic enzymes, and a variety of cytoplasmic proteins lacking an N-terminal secretion signal have been widely described as located at the cell surface in yeast and in mammalian cells, their presence in this external location is still controversial. Here, we report that different experimental approaches (genetics, cellular biology and proteomics) show that yeast enolase can reach the cell surface and describe the protein regions involved in its cell surface targeting. Hybrid enolase truncates, fused at their C terminus with the yeast internal invertase or green fluorescent protein (GFP) as reporter proteins, proved that the 169 N-terminal amino acids are sufficient to target the protein to the cell surface. Furthermore, the enolase-GFP fusion co-localized with a plasma membrane marker. Enolase was also identified among membrane proteins obtained by a purification protocol that includes sodium carbonate to prevent cytoplasmic contamination. These proteins were analyzed by SDS-PAGE, trypsin digestion and LC-MS/MS for peptide identification. Elongation factors, mitochondrial membrane proteins and a mannosyltransferase involved in cell wall mannan biosynthesis were also identified in this fraction.     

Export of major cell surface proteins is blocked in yeast secretory mutants

The transport of newly synthesized proteins to the yeast cell surface has been analyzed by a modification of the technique developed by Kaplan et al. (Kaplan, G., C. Unkeless, and Z.A. Cohn, 1979, Proc. Natl. Acad. Sci. USA, 76:3824-3828). Cells metabolically labeled with (35)SO(4)(2-) are treated with trinitrobenzenesulfonic acid (TNBS) at 0 degrees C under conditions where cell-surface proteins are tagged with trinitrophenol (TNP) but cytoplasmic proteins are not. After fractionation of cells into cell wall, membrane and cytoplasmic samples, and solubilization with SDS, the tagged proteins are immunoprecipitated with anti-TNP antibody and fixed staphylococcus aureus cells. Analysis of the precipitates by SDS gel electrophoresis and fluorography reveals four major protein species in the cell wall (S(1)-S(4)), seven species in the membrane fraction (M(1)-M(7)), and no tagged proteins in the cytoplasmic fraction. Temperature-sensitive mutants defective in secretion of invertase and acid phosphatase (sec mutants; Novick, P., C. Field, and R. Schekman, 1980, Cell, 21:204-215) are also defective in transport of the 11 major cell surface proteins at the nonpermissive temperature (37 degrees C). Export of accumulated proteins is restored in an energy- dependent fashion when secl cells are returned to a permissive temperature (24 degrees C). In wild-type cells the transit time for different surface proteins varies from less than 8 min to about 30 min. The asynchrony is developed at an early stage in the secretory pathway. All of the major cell wall proteins and many of the externally exposed plasma membrane proteins bind to concanavalin A. Inhibition of asparagine-linked glycosylation with tunicamycin does not prevent transport of several surface proteins.     

Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen

Secreted and cell surface proteins play essential roles in numerous essential biological processes in eukaryotic organisms, but are often more difficult to isolate and identify than proteins that are localized in intracellular compartments. However, several high-throughput 'gene-trap' techniques have been developed to characterize these 'secretomes', including the yeast secretion trap (YST) screen. This method involves fusing cDNA libraries from the tissue or cell type of interest to a yeast (Saccharomyces cerevisiae) invertase reporter gene, transforming the resulting fusion library into an invertase-deficient yeast strain and plating the transformants on a medium containing sucrose as the sole carbon source. A yeast cell with a transgene encoding a secreted or cell surface protein can synthesize a secreted invertase fusion protein that can rescue the mutant, and the plasmid DNA can then be sequenced to identify the gene that encodes it. We describe a recently improved version of this screen, which allows the identification of genes encoding secreted proteins in 1-2 months.     

Mating pheromone-induced alteration of cell surface proteins in the heterobasidiomycetous yeast Tremella mesenterica.

Mating pheromone-induced alteration of the cell surface proteins of haploid cells, presumed to play crucial roles in the specific cell-cell interactions during sexual conjugation of Tremella mesenterica , was investigated. Exposed surface proteins were revealed by lactoperoxidase-catalyzed iodination in combination with polyacrylamide gel electrophoresis and autoradiography. From comparison of the molecular species of 125I-labeled surface proteins of the vegetative and the gamete (mating pheromone-treated) cells of the two compatible mating types (ab and AB), it was suggested that a striking change in cell surface structure occurs during the differentiation; although labeled protein species of the vegetative cells of the two mating types were indistinguishable, several new species, both mating type specific and nonspecific, appeared in the gamete cells. Turnover of the labeled proteins of the vegetative cells was negligible, whereas that of the gamete cells was rapid with release of low-molecular-weight labeled proteins in the medium. A role for the labeled surface proteins of the gamete cells in the cell-cell interactions during sexual conjugation was suggested by the following: the surface changes were induced by mating pheromone; the labeled proteins were preferentially localized on the surface of the mating tube; the labeled species appeared sequentially during the differentiation; and mating type-specific species were present in both mating types.     

Effect of some proteins on the yeast cell membrane

Yeast cells, Candida utilis, in water suspension and in the absence of electrolytes were found to be very sensitive to several proteins of moderate size, including ribonuclease, protamine, lysozyme, bovine serum albumin, cytochrome c, and myoglobin. Viability ceases rapidly, and ultraviolet-absorbing compounds (260 mmu) and the amino acid pool are released into the medium. The ultraviolet-absorbing material appears to be the nucleotide and coenzyme fraction usually extracted by 0.2 n perchloric acid at low temperature. The ribonucleic acid fraction remains in the cell ghosts and can be released by ribonuclease. The enzymatic properties of some of these proteins have no relation to their damaging effect on the cell membrane. Poly-l-lysine shows the same activity.     

Chemoselective surface immobilization of proteins through a cleavable peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.     

木糖异构酶在酿酒酵母细胞表面的展示

将来源于嗜热细菌Thermus thermophilus的木糖异构酶基因xylA,与酿酒酵母(Sac-charomyces cerevisiae)a-凝集素表面展示载体pYD1的Aga2p亚基C端序列融合。编码融合蛋白的基因序列前接上半乳糖诱导型启动子。用LiAc完整细胞法转化酿酒酵母EBY100。含重组质粒的菌株EBY100/pYD-xylA经半乳糖诱导表达外源融合蛋白,免疫荧光显微镜结果显示外源蛋白被锚定在细胞壁上,木糖异构酶活性测定结果表明,细胞壁上酶活测定值为1.52U,木糖异构酶在酿酒酵母细胞壁上得到活性表达。     

Oriented immobilization of proteins on hydroxyapatite surface using bifunctional bisphosphonates as linkers

Oriented immobilization of proteins is an important step in creating protein-based functional materials. In this study, a method was developed to orient proteins on hydroxyapatite (HA) surfaces, a widely used bone implant material, to improve protein bioactivity by employing enhanced green fluorescent protein (EGFP) and β-lactamase as model proteins. These proteins have a serine or threonine at their N-terminus that was oxidized with periodate to obtain a single aldehyde group at the same location, which can be used for the site-specific immobilization of the protein. The HA surface was modified with bifunctional hydrazine bisphosphonates (HBPs) of various length and lipophilicity. The number of functional groups on the HBP-modified HA surface, determined by a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, was found to be 2.8 × 10(-5) mol/mg of HA and unaffected by the length of HBPs. The oxidized proteins were immobilized on the HBP-modified HA surface in an oriented manner through formation of a hydrazone bond. The relative protein immobilization amounts through various HBPs were determined by fluorescence and bicinchoninic acid (BCA) assay and showed no significant effect by length and lipophilicity of HBPs. The relative amount of HBP-immobilized EGFP was found to be 10-15 fold that of adsorbed EGFP, whereas the relative amount of β-lactamase immobilized through HBPs (2, 3, 4, 6, and 7) was not significantly different than adsorbed β-lactamase. The enzymatic activity of HBP-immobilized β-lactamase was measured with cefazolin as substrate, and it was found that the catalytic efficiency of HBP-immobilized β-lactamase improved 2-5 fold over adsorbed β-lactamase. The results obtained demonstrate the feasibility of our oriented immobilization approach and showed an increased activity of the oriented proteins in comparison with adsorbed proteins on the same hydroxyapatite surface matrix.     

Dual display of proteins on the yeast cell surface simplifies quantification of binding interactions and enzymatic bioconjugation reactions

Yeast surface display, a well‐established technology for protein analysis and engineering, involves expressing a protein of interest as a genetic fusion to either the N‐ or C‐terminus of the yeast Aga2p mating protein. Historically, yeast‐displayed protein variants are flanked by peptide epitope tags that enable flow cytometric measurement of construct expression using fluorescent primary or secondary antibodies. Here, we built upon this technology to develop a new yeast display strategy that comprises fusion of two different proteins to Aga2p, one to the N‐terminus and one to the C‐terminus. This approach allows an antibody fragment, ligand, or receptor to be directly coupled to expression of a fluorescent protein readout, eliminating the need for antibody‐staining of epitope tags to quantify yeast protein expression levels. We show that this system simplifies quantification of protein‐protein binding interactions measured on the yeast cell surface. Moreover, we show that this system facilitates co‐expression of a bioconjugation enzyme and its corresponding peptide substrate on the same Aga2p construct, enabling enzyme expression and catalytic activity to be measured on the surface of yeast.     

Zeta potential of yeast cells: application in cell immobilization

The zeta potential of Saccharomyces cerevisiae cells has been studied. Zeta potential was measured by microelectrophoresis with a Laser Zee meter model 500. Haploid (a and α) and diploid cells were tested and their zeta potential was found to be comparable. The influence of agents such as CaCl₂, NaCl, carboxymethylcellulose, Al₂(SO₄)₃ and cationic starch, on the zeta potential of cells has been studied. Moreover, zeta potential measurement has been used for improvement of cell immobilization by adhesion on sawdust.     

Spacer-mediated display of active lipase on the yeast cell surface

We have constructed a Saccharomyces cerevisiae strain displaying an active lipase on the cell surface by cell surface engineering. The gene encoding Rhizopus oryzae lipase (ROL) was fused with the genes encoding the pre-alpha-factor leader sequence and the C-terminal half of alpha-agglutinin including the glycosylphosphatidylinositol-anchor attachment signal. The constructed gene was overexpressed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance lipase activity by preserving the conformation of the active site near the C-terminal portion. Localization of the expressed ROL on the cell surface was confirmed by immunofluorescence microscopy. The ROL displayed on the yeast cell wall exhibited activity toward soluble 2,3-dimercaptopropan-1-ol tributyl ester (BALB) and insoluble triolein. The insertion of linker peptides effected the activity towards BALB, thereby demonstrating that the optimal length of linker peptides was present. The activity towards triolein was higher in lipases with longer linker peptides. ROL displayed on the cell wall exhibited a comparable and/or higher activity towards triolein than the secreted form of the enzyme. This is the first report of an active lipase displayed on the cell surface. Furthermore, insertion of a linker peptide of the appropriate length as a spacer may be an improved method to effectively display enzymes, especially those having the active region at the C-terminal portion, on the cell surface.     

The effect of yeast cell immobilization on the proportion of selected by-products of ethanol fermentation

Determination was made of the proportion of selected by-products (acetaldehyde, ethyl acetate, methanol, propanol, isobutanol, 2-methyl-butanol, 3-methyl-butanol) of batch and continuous ethanol fermentation carried out with the use of yeastSaccharomyces cerevisiae, strain 0–11, cells immobilized by adsorption on selected carriers (foamed polystyrene, bone shot, beech wood chips, porous glass) as well as by entrapping in calcium alginate and calcium pectinate gel.     

Development of ultraporous fired bricks as support for yeast cell immobilization

Ultraporous fired bricks (porosities from 56 to 72%) were developed from materials locally available in Nigeria. The grog particle size was used to modulate the porosity of the bricks. The porous bricks produced were then employed as supports for the immobilization of a yeast strain isolated from a local alcoholic beverage, palm wine. The influence of a brick's porosity and particle size on the cell-loading capacity and cell growth inside the fired brick support were studied. The study revealed that a brick's porosity varied linearly with the mean particle size of the grog, increasing from a porosity of 56% at a particle size of 0.805 mm to 72% at a particle size of 0.075 mm. Cell saturation of the surface area available within the support matrix was completed within four hours of contact between the cell and the adsorbing surface especially for the most porous samples. Cell growth was therefore not observed in such cases; however, the less porous samples supported some cell growth upon incubation. Cell holdup was also observed to increase exponentially when either the porosity was increased or the particle size was decreased. The influence of particle size, however, became insignificant at very high porosities.     

Synthesis of ribosomal proteins during the yeast cell cycle

The synthesis of ribosomal proteins during the cell division cycle of Saccharomyces cerevisiae has been examined. A technique was utilized whereby cells in unique phases of the cell cycle were selected from an asynchronous culture after the period of pulse labeling. Some of the proteins of the small and large ribosomal subunits were synthesized continuously throughout the cell cycle and there was no evidence of discontinuous synthesis for any of the ribosomal proteins.