Display of adenoregulin with a novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> cell surface display system

Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo 1p with its own secretion signal sequence or the α-factor secretion signal sequence, a polyhistidine (6×His) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.     

Construction of a Novel <Emphasis Type="Italic">Pichia pastoris</Emphasis> Cell-Surface Display System Based on the Cell Wall Protein Pir1

A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study. The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface. The fluorescence intensity of the whole yeast was measured by spectrofluorometer. Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment. The results indicate that a Pir1-based system can express proteins on the surface of P. pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall. The repetitive sequences of Pir1 are required for cell wall retention, and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P. pastoris.     

Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP ₆₃₀ ) or a short-TIP1 fragment (ScTIP ₁₂₀ ) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody, suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface display of foreign proteins in P. pastoris.     

Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines

Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.     

Expression and localization of recombinant human B2 receptors in the methylotrophic yeast Pichia pastoris

The human bradykinin B₂ receptor (B₂R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast Pichia pastoris. In the expression vector, B₂R gene was driven under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and Western blot analysis, it was proved that B₂R recombinant receptor proteins were expressed at a high level in the yeast. Furthermore, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B₂R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy. The text was submitted by the authors in English.     

Flow cytometry‐based enrichment for cell shape mutants identifies multiple genes that influence Helicobacter pylori morphology

The helical cell shape of Helicobacter pylori is highly conserved and contributes to its ability to swim through and colonize the viscous gastric mucus layer. A multi‐faceted peptidoglycan (PG) modification programme involving four recently characterized peptidases and two accessory proteins is essential for maintaining H. pylori's helicity. To expedite identification of additional shape‐determining genes, we employed flow cytometry with fluorescence‐activated cell sorting (FACS) to enrich a transposon library for bacterial cells with altered light scattering profiles that correlate with perturbed cell morphology. After a single round of sorting, 15% of our clones exhibited a stable cell shape defect, reflecting 37‐fold enrichment. Sorted clones with straight rod morphology contained insertions in known PG peptidases, as well as an insertion in csd6, which we demonstrated has ld ‐carboxypeptidase activity and cleaves monomeric tetrapeptides in the PG sacculus, yielding tripeptides. Other mutants had only slight changes in helicity due to insertions in genes encoding MviN/MurJ, a protein possibly involved in initiating PG synthesis, and the hypothetical protein HPG27_782. Our findings demonstrate FACS robustly detects perturbations of bacterial cell shape and identify additional PG peptide modifications associated with helical cell shape in H. pylori.     

Construction and characterization of normalized cDNA library of maize inbred MO17 from multiple tissues and developmental stages

A comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10 830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarray was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using the BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate genes, and developing microarrays in maize genomics research.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 198–206.Original English Text Copyright © 2005 by Z. Zhang, F. Zhang, Tang, Pi, Zheng.This article was submitted by the authors in English.     

Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains

Expression of proteins on the surface of yeasts has a wide range of applications in biotechnology, such as directed evolution of proteins for increased affinity and thermal stability, screening of antibody libraries, epitope mapping, and use as whole-cell biocatalysts. However, hyperglycosylation can interfere with overall protein accessibility on the surface. Therefore, the less elaborate hyperglycosylation in wild type Pichia pastoris and the availability of glycoengineered strains make this yeast an excellent alternative for surface display of glycoproteins. Here, we report the implementation of the well-established a-agglutinin-based yeast surface display technology in P. pastoris. Four heterologous proteins were expressed on the surface of a wild type and a glycoengineered strain. Surface display levels were monitored by Western blot, immunofluorescence microscopy, and FACS analysis. The availability of glycoengineered strains makes P. pastoris an excellent alternative for surface display of glycoproteins and paves the way for new applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cell‐free translational screening of an expression sequence tag library of Clonorchis sinensis for novel antigen discovery

The rapidly evolving cloning and sequencing technologies have enabled understanding of genomic structure of parasite genomes, opening up new ways of combatting parasite‐related diseases. To make the most of the exponentially accumulating genomic data, however, it is crucial to analyze the proteins encoded by these genomic sequences. In this study, we adopted an engineered cell‐free protein synthesis system for large‐scale expression screening of an expression sequence tag (EST) library of Clonorchis sinensis to identify potential antigens that can be used for diagnosis and treatment of clonorchiasis. To allow high‐throughput expression and identification of individual genes comprising the library, a cell‐free synthesis reaction was designed such that both the template DNA and the expressed proteins were co‐immobilized on the same microbeads, leading to microbead‐based linkage of the genotype and phenotype. This reaction configuration allowed streamlined expression, recovery, and analysis of proteins. This approach enabled us to identify 21 antigenic proteins. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:832–837, 2017     

MOLECULAR CHARACTERIZATION OF A PHOSPHATE‐REGULATED CELL‐SURFACE PROTEIN FROM THE COCCOLITHOPHORID,EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE)1

Emiliania huxleyi (Lohmann) Hay et Mohler is a cosmopolitan coccolithophorid that is known to be an excellent competitor for phosphate. A previous survey of cell‐surface proteins induced by phosphorus limitation in strain CCMP 374 yielded three abundant proteins. Using CCMP 1516, the strain chosen for genome sequence determination, we report the cDNA, genomic, and amino acid sequence of one cell‐surface phosphorus‐limitation induced protein and evidence that a second protein is highly similar. The introns within the genomic DNA encoding this cell‐surface protein as well as those defined by other phosphate‐regulated expressed sequence tags are analyzed. As these proteins are the most abundant cell‐surface proteins present under phosphorus limitation, they likely have a role in the ability of this organism to compete for phosphate.