Blending of selected yeast extract and peptone for inducible and constitutive protein production in Escherichia coli using the pET system.

pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl β-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene.

The presence of an intron relieves gene repression caused by promoter-proximal four-bp specific sequences in yeast.

Introns can enhance gene expression in eukaryotic cells in a process called intron-mediated enhancement (IME). The levels of enhancement are affected not only by the intron sequence but also by coding sequences (CDSs). However, the parts of CDSs responsible for mediating IME have not yet been identified.

Triple gene expressions in yeast, Escherichia coli, and mammalian cells by transferring DNA fragments amplified from a mother yeast expression plasmid.

Escherichia coli, Saccharomyces cerevisiae, and mammalian culture cells are standard host organisms for genetic engineering and research, thus various plasmid vectors have been developed. However, the vectors are designed only for a single host owing to their host-specific genetic elements such as promoters and selection markers. In this study, we developed a yeast expression plasmid that enables the expression of the same gene in E.

Identification of essential intron sequences that enhance gene expression independently of splicing in the yeast Saccharomyces cerevisiae.

Gene expression in eukaryotes is enhanced by the presence of introns in a process known as intron-mediated enhancement (IME), but its mechanism remains unclear. In Saccharomyces cerevisiae, sequences at the 5'-splice sites (SS) and branch point sites (BPS) are highly conserved compared with other higher eukaryotes. Here, the minimum intron sequence essential for IME was investigated using various short introns and a yeast codon-optimized luciferase gene as an IME model.

Various short autonomously replicating sequences from the yeast seemingly without canonical consensus.

Eukaryotic autonomously replicating sequences (ARSs) are composed of three domains, A, B, and C. Domain A is comprised of an ARS consensus sequence (ACS), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. In and ARS101, the ACS is commonly composed of 11 bp, 5'-(A/T)AAA(C/T)ATAAA(A/T)-3'.

Inhibition of Distinct Proline- or -Acetylglucosamine-Induced Hyphal Formation Pathways by Proline Analogs in .

undergoes a yeast-to-hyphal transition that has been recognized as a virulence property as well as a turning point leading to biofilm formation associated with candidiasis. It is known that yeast-to-hyphal transition is induced under complex environmental conditions including temperature (above 35°C), pH (greater than 6.5), CO, -acetylglucosamine (GlcNAc), amino acids, RPMI-1640 synthetic culture medium, and blood serum.

Anoxia-induced mitophagy in the yeast Kluyveromyces marxianus.

Kluyveromyces marxianus is a thermotolerant, ethanol-producing yeast that requires oxygen for efficient ethanol fermentation. Under anaerobic conditions, glucose consumption and ethanol production are retarded, suggesting that oxygen affects the metabolic state of K. marxianus.

Autophagy regulates levels of tumor suppressor enzyme protein phosphatase 6.

Protein phosphatase 6 (PP6) is an essential serine/threonine protein phosphatase that acts as an important tumor suppressor. However, increased protein levels of PP6 have been observed in some cancer types, and they correlate with poor prognosis in glioblastoma. This raises a question about how PP6 protein levels are regulated in normal and transformed cells.

YHp as a highly stable, hyper-copy, hyper-expression plasmid constructed using a full 2-μm circle sequence in cir strains of Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, the yeast episomal plasmid (YEp), containing a partial sequence from a natural 2-μm plasmid, has been frequently used to induce high levels of gene expression. In this study, we used Japanese sake yeast natural cir strain as a host for constructing an entire 2-μm plasmid with an expression construct using the three-fragment gap-repair method without Escherichia coli manipulation. The 2-μm plasmid contains two long inverted repeats, which is problematic for the amplification by polymerase chain reaction.

Identification and Mutational Analysis of Escherichia coli Sorbitol-Enhanced Glucose-Repressed srlA Promoter Expressed in LB Medium by Using Homologous Recombination and One-Round PCR Products.

Escherichia coli has been used for recombinant protein production for many years. However, no native E. coli promoters have been found for constitutive expression in LB medium.

Enhanced production of extracellular inulinase by the yeast Kluyveromyces marxianus in xylose catabolic state.

The production of extracellular proteins by the thermotolerant yeast Kluyveromyces marxianus, which utilizes various sugars, was investigated using media containing sugars such as glucose, galactose, and xylose. SDS-PAGE analysis of culture supernatants revealed abundant production of an extracellular protein when cells were grown in xylose medium. The N-terminal sequence of the extracellular protein was identical to a part of the inulinase encoded by INU1 in the genome.

Enzymatic Hydrolysis of Black Liquor Xylan by a Novel Xylose-Tolerant, Thermostable β-Xylosidase from a Tropical Strain of Aureobasidium pullulans CBS 135684.

From three cell-associated β-xylosidases produced by Aureobasidium pullulans CBS 135684, the principal enzyme was enriched to apparent homogeneity and found to be active at high temperatures (60-70 °C) over a pH range of 5-9 with a specific activity of 163.3 units (U) mg. The enzyme was thermostable, retaining over 80% of its initial activity after a 12-h incubation at 60 °C, with half-lives of 38, 22, and 10 h at 60, 65, and 70 °C, respectively.

Polymorphisms in the LAC12 gene explain lactose utilisation variability in Kluyveromyces marxianus strains.

Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide.

5´-UTR introns enhance protein expression in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae is one of the most suitable microorganisms for recombinant protein production. To enhance protein production, various expression systems have been intensively studied. However, the effect of introns on protein expression has not been examined deeply in S.

Selection of Error-Less Synthetic Genes in Yeast.

Conventional gene synthesis is usually accompanied by sequence errors, which are often deletions derived from chemically synthesized oligonucleotides. Such deletions lead to frame shifts and mostly result in premature translational terminations. Therefore, in-frame fusion of a marker gene to the downstream of a synthetic gene is an effective strategy to select for frame-shift-free synthetic genes.

Genomic analyses of thermotolerant microorganisms used for high-temperature fermentations.

Environmental adaptation is considered as one of the most challenging subjects in biology to understand evolutionary or ecological diversification processes and in biotechnology to obtain useful microbial strains. Temperature is one of the important environmental stresses; however, microbial adaptation to higher temperatures has not been studied extensively. For industrial purposes, the use of thermally adapted strains is important, not only to reduce the cooling expenses of the fermentation system, but also to protect fermentation production from accidental failure of thermal management.

The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy.

Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses.

End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies.

Gene expression analysis using strains constructed by NHEJ-mediated one-step promoter cloning in the yeast Kluyveromyces marxianus.

Gene expression analysis provides valuable information to evaluate cellular state. Unlike quantitative mRNA analysis techniques like reverse-transcription PCR and microarray, expression analysis using a reporter gene has not been commonly used for multiple-gene analysis, probably due to the difficulty in preparing multiple reporter-gene constructs. To circumvent this problem, we developed a novel one-step reporter-gene construction system mediated by non-homologous end joining (NHEJ) in the yeast Kluyveromyces marxianus.

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells.

Escherichia coli plasmids are commonly used for gene expression experiments in mammalian cells, while PCR-amplified DNAs are rarely used even though PCR is a much faster and easier method to construct recombinant DNAs. One difficulty may be the limited amount of DNA produced by PCR. For direct utilization of PCR-amplified DNA in transfection experiments, efficient transfection with a smaller amount of DNA should be attained.

Mutant screening for oncogenes of Ewing's sarcoma using yeast.

Many fusion genes, which are the result of chromosomal translocation and work as an oncogene, have been recently identified, but their mode of actions is still unclear. Here, we performed a yeast mutant screening for oncogenes of Ewing's sarcoma to easily identify essential regions responsible for fusion protein functions using a yeast genetic system. Three kinds of oncogenes including EWS/FLI1, EWS/ERG, and EWS/E1AF exhibited growth inhibition in yeast.

Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

Background: Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences.

Screening of accurate clones for gene synthesis in yeast.

Methods for error-less gene synthesis are desired because synthesized genes often contain mutations. By cloning PCR-assembled oligonucleotide fragments fused to a selection marker in yeast, we developed a novel method to screen accurate clones in gene synthesis. As a model case, the 555-bp luciferase gene from Gaussia princeps (GLuc) was synthesized to contain yeast-optimized codons (called yGLuc hereafter).

Structural basis of starvation-induced assembly of the autophagy initiation complex.

Assembly of the preautophagosomal structure (PAS) is essential for autophagy initiation in yeast. Starvation-induced dephosphorylation of Atg13 is required for the formation of the Atg1-Atg13-Atg17-Atg29-Atg31 complex (Atg1 complex), a prerequisite for PAS assembly. However, molecular details underlying these events have not been established.

Minimum GC-rich sequences for overlap extension PCR and primer annealing.

PCR is a common method to produce desired DNA fragments from templates. The oligonucleotide primers used for PCR must contain annealing sequences that are usually 20-30 nucleotides long and identical to a part of template DNA. However, primers often contain additional sequences at their 5' ends, which are restriction enzyme sites, recombination targeting sequences, or overlap sequences for fusion PCR.