A new type of gene‐disruption cassette with a rescue gene for Pichia pastoris

Pichia pastoris has been used for the production of many recombinant proteins, and many useful mutant strains have been created. However, the efficiency of mutant isolation by gene‐targeting is usually low and the procedure is difficult for those inexperienced in yeast genetics. In order to overcome these issues, we developed a new gene‐disruption system with a rescue gene using an inducible Cre/mutant–loxP system. With only short homology regions, the gene‐disruption cassette of the system replaces its target–gene locus containing a mutation with a compensatory rescue gene. As the cassette contains the AOX1 promoter‐driven Cre gene, when targeted strains are grown on media containing methanol, the DNA fragment, i.e., the marker, rescue and Cre genes, between the mutant‐loxP sequences in the cassette is excised, leaving only the remaining mutant‐loxP sequence in the genome, and consequently a target gene‐disrupted mutant can be isolated. The system was initially validated on ADE2 gene disruption, where the disruption can easily be detected by color‐change of the colonies. Then, the system was applied for knocking‐out URA3 and OCH1 genes, reported to be difficult to accomplish by conventional gene‐targeting methods. All three gene‐disruption cassettes with their rescue genes replaced their target genes, and the Cre/mutant–loxP system worked well to successfully isolate their knock‐out mutants. This study identified a new gene‐disruption system that could be used to effectively and strategically knock out genes of interest, especially whose deletion is detrimental to growth, without using special strains, e.g., deficient in nonhomologous end‐joining, in P. pastoris. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1201–1208, 2017     

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.     

A wheat embryo cell‐free protein synthesis system not requiring an exogenous supply of GTP

Most in vitro protein synthesis systems require a supply of GTP for the formation of translation initiation complexes, with two GTP molecules per amino acid needed as an energy source for a peptide elongation reaction. In order to optimize protein synthesis reactions in a continuous‐flow wheat embryo cell‐free system, we have examined the influence of adding GTP and found that the system does not require any supply of GTP. We report here the preparation of a wheat embryo extract from which endogenous GTP was removed by gel filtration, and the influence of adding GTP to the system on protein synthesis reactions. Using Green Fluorescent Protein (GFP) as a reporter, higher levels of production were observed at lower concentrations of GTP, with the optimal level of production obtained with no supply of GTP. A HPLC‐based analysis of the extract and the translation mixture containing only ATP as an energy source revealed that GTP was not detectable in the extract, however, 35 μM of GTP was found in the translation mixture. This result suggests that GTP could be generated from other compounds, such as GDP and GMP, using ATP. A similar experiment with a C‐terminally truncated form of human protein tyrosine phosphatase 1B (hPTP1B_1‐320) gave almost the same result. The wheat embryo cell‐free translation system worked most efficiently without exogenous GTP, producing 3.5 mg/mL of translation mixture over a 48‐h period at 26°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Kinetic analyses for bindings of concanavalin A to dispersed and condensed mannose surfaces on a quartz crystal microbalance

A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (K_a) and binding and dissociation rate constants (k_on and k_off) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as K_a = (4 ± 1) × 10⁶ M⁻¹, k_on = (4 ± 1) × 10⁴ M⁻¹ s⁻¹, and k_off = (12 ± 2) × 10^–3 s⁻¹. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as K_a = (14 ± 2) × 10⁶ M⁻¹, k_on = (14 ± 2) × 10⁴ M⁻¹ s⁻¹, and k_off = (7 ± 2) × 10^–3 s⁻¹. Thus, K_a for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (k_on) and a three times smaller dissociation rate constant (k_off). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface.     

MGL2+ Dermal Dendritic Cells Are Sufficient to Initiate Contact Hypersensitivity In Vivo

Background

Dendritic cells (DCs) are the most potent antigen-presenting cells in the mammalian immune system. In the skin, epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) survey for invasive pathogens and present antigens to T cells after migration to the cutaneous lymph nodes (LNs). So far, functional and phenotypic differences between these two DC subsets remain unclear due to lack of markers to identify DDCs.

Methodology/Principal Findings

In the present report, we demonstrated that macrophage galactose-type C-type lectin (MGL) 2 was exclusively expressed in the DDC subset in the skin-to-LN immune system. In the skin, MGL2 was expressed on the majority (about 88%) of MHCII⁺CD11c⁺ cells in the dermis. In the cutaneous LN, MGL2 expression was restricted to B220⁻CD8α^loCD11b⁺CD11c⁺MHCII^hi tissue-derived DC. MGL2⁺DDC migrated from the dermis into the draining LNs within 24 h after skin sensitization with FITC. Distinct from LCs, MGL2⁺DDCs localized near the high endothelial venules in the outer T cell cortex. In FITC-induced contact hypersensitivity (CHS), adoptive transfer of FITC⁺MGL2⁺DDCs, but not FITC⁺MGL2⁻DCs into naive mice resulted in the induction of FITC-specific ear swelling, indicating that DDCs played a key role in initiation of immune responses in the skin.

Conclusions/Significance

These results demonstrated the availability of MGL2 as a novel marker for DDCs and suggested the contribution of MGL2⁺ DDCs for initiating CHS.     

Tyro3 family-mediated cell entry of Ebola and Marburg viruses

Filoviruses, represented by the genera Ebolavirus and Marburgvirus, cause a lethal hemorrhagic fever in humans and in nonhuman primates. Although filovirus can replicate in various tissues or cell types in these animals, the molecular mechanisms of its broad tropism remain poorly understood. Here we show the involvement of members of the Tyro3 receptor tyrosine kinase family-Axl, Dtk, and Mer-in cell entry of filoviruses. Ectopic expression of these family members in lymphoid cells, which otherwise are highly resistant to filovirus infection, enhanced infection by pseudotype viruses carrying filovirus glycoproteins on their envelopes. This enhancement was reduced by antibodies to Tyro3 family members, Gas6 ligand, or soluble ectodomains of the members. Live Ebola viruses infected both Axl- and Dtk-expressing cells more efficiently than control cells. Antibody to Axl inhibited infection of pseudotype viruses in a number of Axl-positive cell lines. These results implicate each Tyro3 family member as a cell entry factor in filovirus infection.     

Biochemical characterization of the very long-chain fatty acid elongase ELOVL7

Very long-chain fatty acids (VLCFAs) have a variety of physiological functions and are related to numerous disorders. The key step of VLCFA elongation is catalyzed by members of the elongase family, ELOVLs. Mammals have seven ELOVLs (ELOVL1-7), yet none of them has been purified and analyzed. In the presented study we purified ELOVL7 and measured its activity by reconstituting it into proteoliposomes. Purified ELOVL7 exhibited high activity toward acyl-CoAs with C18 carbon chain length. The calculated K(m) values toward C18:3(n-3)-CoA and malonyl-CoA were both in the μM range. We also found that progression of the VLCFA cycle enhances ELOVL7 activity.     

Cysteine-to-serine shuffling using a Saccharomyces cerevisiae expression system improves protein secretion: case of a nonglycosylated mutant of miraculin, a taste-modifying protein

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.     

The relationship between muscle deoxygenation and activation in different muscles of the quadriceps during cycle ramp exercise

The relationship between muscle deoxygenation and activation was examined in three different muscles of the quadriceps during cycling ramp exercise. Seven young male adults (24 ± 3 yr; mean ± SD) pedaled at 60 rpm to exhaustion, with a work rate (WR) increase of 20 W/min. Pulmonary oxygen uptake was measured breath-by-breath, while muscle deoxygenation (HHb) and activity were measured by time-resolved near-infrared spectroscopy (NIRS) and surface electromyography (EMG), respectively, at the vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM). Muscle deoxygenation was corrected for adipose tissue thickness and normalized to the amplitude of the HHb response, while EMG signals were integrated (iEMG) and normalized to the maximum iEMG determined from maximal voluntary contractions. Muscle deoxygenation and activation were then plotted as a percentage of maximal work rate (%WR(max)). The HHb response for all three muscle groups was fitted by a sigmoid function, which was determined as the best fitting model. The c/d parameter for the sigmoid fit (representing the %WR(max) at 50% of the total amplitude of the HHb response) was similar between VL (47 ± 12% WR(max)) and VM (43 ± 11% WR(max)), yet greater (P < 0.05) for RF (65 ± 13% WR(max)), demonstrating a "right shift" of the HHb response compared with VL and VM. The iEMG also showed that muscle activation of the RF muscle was lower (P < 0.05) compared with VL and VM throughout the majority of the ramp exercise, which may explain the different HHb response in RF. Therefore, these data suggest that the sigmoid function can be used to model the HHb response in different muscles of the quadriceps; however, simultaneous measures of muscle activation are also needed for the HHb response to be properly interpreted during cycle ramp exercise.