The structure and stability analysis of the pea seed legumin glycosylated by oligochitosan.

BACKGROUND: The functionality of pea proteins is relatively weak relative to that of soybean proteins, which limits the application of pea proteins in food and nutritional applications. Glycosylation is a promising approach to influence the protein structure and in turn change the functional properties of pea proteins. RESULTS: In this study, the effect of transglutaminase-induced oligochitosan glycosylation on the structural and functional properties of pea seed legumin was studied. Different oligochitosan-modified legumin complexes (OLCs) were prepared by applying different molar ratios of legumin to oligochitosan (1:1 to 1:4) induced by transglutaminase (10 U g-1 protein). Results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), glucosamine, and free amino analysis showed that the legumin could be covalently bonded with the oligochitosan and were influenced by the applying dose of the oligochitosan. Infrared spectroscopy, fluorescence, and scanning electron microscopy analysis indicated that the structure of the different OLC samples could be changed to different extents. Moreover, although the emulsifying activity decreased, the emulsification stability, thermal stability, and in vitro digestive stability of the OLCs were remarkably improved relative to that of the untreated legumin. CONCLUSION: Oligochitosan glycosylation could change the structure of the legumin and consequently improve its emulsification stability, thermal stability, and in vitro digestive stability. This study will facilitate the legumin functionalization by the glycosylation approach to fabricate protein-oligochitosan complex for potential food and nutritional applications. © 2020 Society of Chemical Industry.

Development of vaccines for prevention of peste-des-petits-ruminants virus infection.

Peste des petits ruminants (PPR) is a highly contagious and fatal disease of small ruminants, particularly sheep and goats. This disease leads to high morbidity and mortality of small ruminants, thus resulting in devastating economic loss to the livestock industry globally. The severe disease impact has prompted the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (OIE) to develop a global strategy for the control and eradication of PPR by 2030. Over the past decades, the control of PPR is mainly achieved through vaccinating the animals with live-attenuated vaccines, e.g., rinderpest vaccines. As a closely related disease to PPR of large ruminants, rinderpest was eradicated in 2011 and its vaccines subsequently got banned in order to keep rinderpest-free zones. Consequently, it is desirable to develop homologous PPR vaccines to control the disease. The present review summarizes the objectives of PPR control and eradication by focusing on the homologous PPR vaccines.

Expression, purification and characterization of a recombinant antimicrobial peptide Hispidalin in Pichia pastoris.

Hispidalin is a novel antimicrobial peptide isolated from the seeds of Benincasa hispida and is reported to have broad antimicrobial activity against various bacterial and fungal pathogens. To produce significant amounts of Hispidalin, a recombinant Hispidalin with an N-terminal 6 × His tag and an enterokinase sequence, for the first time, was successfully expressed in Escherichia coli or Pichia pastoris cell factory. Results showed that the E. coli-derived recombinant Hispidalin did not show any antimicrobial activity against all the tested strains, whereas the P. pastoris-derived recombinant Hispidalin (rHispidalin) showed a broad antibacterial spectrum against five pathogenic bacteria of both Gram-negative and Gram-positive. rHispidalin also has bactericidal activity and completely killed all of the Staphylococcus aureus within 40 min. Additionally, rHispidalin showed a broad range of thermostability and pH stability, and a hemolytic activity of less than 2% even at a concentration of 300 µg/ml; it was resistant to trypsin and proteinase K, but was moderately sensitive to pepsin and papain. Moreover, rHispidalin effectively permeabilized the cytoplasmic membrane and disrupted the morphology of targeted bacterial cells. After an initial optimization was performed, the amount of rHispidalin accumulation could reach as high as 98.6 µg/ml. These results indicate that Hispidalin could be produced on a large scale by P. pastoris and has a great potential to be utilized as a new antibacterial agent for further development.

Ornithine decarboxylase is involved in methyl jasmonate-regulated postharvest quality retention in button mushrooms (Agaricus bisporus).

BACKGROUND: In the present study, we investigated the role of ornithine decarboxylase (ODC) in the methyl jasmonate (MeJA)-regulated postharvest quality maintenance of Agaricus bisporus (J. E. Kange) Imbach button mushrooms by pretreating mushrooms with a specific irreversible inhibitor called α-difluoromethylornithine (DFMO) before exposure to MeJA vapor. RESULTS: Mushrooms were treated with 0 or 100 µmol L-1 MeJA or a combination of 120 µmol L-1 DFMO and 100 µmol L-1 MeJA, respectively, before storage at 4 °C for 21 days. Treatment with MeJA alone induced the increase in ODC activity whereas this effect was greatly suppressed by pretreatment with DFMO. α-Difluoromethylornithine strongly attenuated the effect of MeJA on decreasing cap opening, slowing the decline rate of soluble protein and total sugar, and accumulating total phenolics and flavonoids. α-Difluoromethylornithine pretreatment also counteracted the ability of MeJA to inhibit polyphenol oxidase and lipoxygenase activities, and malondialdehyde production, and to stimulate superoxide dismutase and catalase activities. It also largely downregulated MeJA-induced accumulation of free putrescine (Put). CONCLUSION: These results reveal that ODC is involved in MeJA-regulated postharvest quality retention of button mushrooms, and this involvement is likely to be associated with Put levels. © 2018 Society of Chemical Industry.

Efficient production of a recombinant Venerupis philippinarum defensin (VpDef) in Pichia pastoris and characterization of its antibacterial activity and stability.

VpDef is a novel defensin isolated from the clam Venerupis philippinarum. Previously it was expressed in Escherichia coli; however, the E. coli-derived recombinant VpDef did not show effective antimicrobial activity against Staphyloccocus aureus or the Gram-negative bacteria tested. As such, the goal of this study was to design, express, and purify a recombinant VpDef (rVpDef) in Pichia pastoris and to determine its antibacterial potency and stability. A 6.9 KDa rVpDef was successfully expressed as a secreted peptide in P. pastoris, and the amount of rVpDef accumulation was shown to reach as high as approximate 60 µg per 1 ml of culture medium only after an initial optimization was performed. The purified rVpDef demonstrated a broad antibacterial spectrum and was active against six typical common bacteria, both Gram-positive and Gram-negative. A minimal inhibition concentration of as low as 50 µg/ml was observed for rVpDef against the growth of E. coli O157 (ATCC 35150). Moreover, rVpDef was tolerant to temperature shock and proteinase digestion and maintained a high stability over a relatively broad pH range. In addition, rVpDef had a low hemolytic activity against rabbit erythrocytes. Taken together, this study demonstrated that rVpDef could be produced in a large-scale manner in P. pastoris and has a good antibacterial activity and suitable stability. This is the first report on heterologous expression of a biologically active VpDef in P. pastoris, supporting its use for both research and application purposes.

Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris.

The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79.6 µg per 1 ml of culture medium. Once the rPaDef peptide was purified to reach a 95.7% purity using one-step nickel affinity chromatography, its strong and concentration-dependent antimicrobial activity was detected to be against a broad-spectrum of bacteria of both Gram-negative and Gram-positive. The growth of these bacterial pathogens was almost completely inhibited when the rPaDef peptide was at a concentration of as low as 90 µg/ml. In summary, our data showed that rPaDef derived from Mexican avocado fruit can be expressed and secreted efficiently when P. pastoris was used as a cell factory. This is the first report on heterologous expression of PaDef in P. pastoris and the approach described holds great promise for antibacterial drug development.

A novel bicistronic expression system composed of the intraflagellar transport protein gene ift25 and FMDV 2A sequence directs robust nuclear gene expression in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii offers a great promise for large-scale production of multiple recombinant proteins of pharmaceutical and industrial interest. However, the nuclear-encoding transgenes usually are expressed at a low level, which severely hampers the use of this alga in molecular farming. In this study, the promoter of the endogenous intraflagellar transport 25 (IFT25) gene of C. reinhardtii was tested for its ability to drive the expression of green fluorescent protein (GFP), which functions as a readout for target gene expression. IFT25 promoter (IFT25P) alone was not able to drive GFP expression to a detectable level. IFT25P, however, can drive robust IFT25-GFP fusion protein expression when the intron-containing IFT25 gene was inserted between IFT25P and GFP cDNA. When an extended version of foot-and-mouth virus 2A protease (2AE) sequence was further inserted between the intron-containing IFT25 gene and the GFP cDNA, discrete GFP protein was observed to release from the IFT25-2AE-GFP polyprotein via 2A self-cleaving with a cleavage efficacy of approximately 99%. The monomer GFP was accumulated to a level of as high as 0.68% of total soluble proteins. To test whether the newly developed bicistronic IFT25P-IFT25-2AE expression system can be used to overexpress heterologous proteins of different origins and sizes, we inserted codon-optimized cDNAs encoding a Trichoderma reesei xylanase1 (25 kDa) and a Lachnospiraceae bacterium ND2006 type V CRISPR-Cas protein LbCpf1 (147 kDa) to the vector and found that the production of xylanase1 and LbCpf1 was as high as 0.69 and 0.49% of total soluble protein. Our result showed that IFT25P-IFT25-2AE system is more efficient to drive nuclear gene expression in C. reinhardtii than other conventionally used promoters, thus representing a novel efficient recombinant protein expression tool and has the potential to be scaled for commercial production of nuclear-encoded recombinant proteins of different sizes and origins in C. reinhardtii.

Expression, purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris.

Mytichitin-A is an antimicrobial peptide isolated from the serum of Mytilus coruscus and is reported to inhibit bacterial growth as tested on several Gram-positive bacteria. To produce large quantity of Mytichitin-A to further investigate its biological activity, nucleotide sequence encoding a recombinant 6 × His-Mytichitin-A (rMytichitin-A) peptide was synthesized and inserted into the inducible yeast expression vector pPICZαA. With the availability of such an expression vector called pPICZαA-Mytichitin-A, we transformed Pichia pastoris GS115 cells with a SacI-linearized pPICZαA-Mytichitin-A by electroporation. Transgenic strains secreting rMytichitin-A with a molecular weight of approximate 10 KDa as expected were obtained. The optimal culture condition for rMytichitin-A expression was determined to be 1.0% methanol induction, 96 h incubation at 28 °C and the amount of rMytichitin-A reached 45.5 µg/ml. The percentage of rMytichitin-A was estimated to be 73.6% of the total protein. After rMytichitin-A was purified using nickel ions affinity chromatography, approximate 9.1 mg pure rMytichitin-A was obtained from 500 ml of cell culture medium with 97.8% purity. More importantly, both the culture supernatant and purified rMytichitin-A inhibited the growth of Gram-positive bacteria, especially Staphylococcus aureus and Bacillus subtilis with a minimum inhibition concentration of as low as 31 and 48 µg/ml, respectively. Differently from the native protein, however, the rMytichitin-A is not active against Gram-negative bacteria. Taken together, this is the first report on the heterologous expression of Mytichitin-A in P. pastoris. Our study showed that P. pastoris is an effective expression system for producing large quantities of biologically active Mytichitin-A for both research and application purposes.

Isolation of high quality and yield of RNA from Agaricus bisporus with a simple, inexpensive and reliable method.

A method for isolating high purity and quantity RNA from Agaricus bisporus which is rich in proteins, carbohydrate, fiber and secondary metabolites, is described. RNA was extracted from mycelium, primordia, sporophores at two development stages and two post-harvest storage stages as well as from pileipellis, inner cap, gill and stipe of the mature sporophore. The A(260)/A(230) and A(260)/A(280) ratios of isolated RNA from fruiting bodies were both ~2 and the yield was about 200 µg/g fresh wt (FW). The yield of RNA from mycelium was approx. 100 µg/g FW. High quality RNA was also extracted from fruiting body tissues of Lentinus edodes, Pleurotus ostreatus, Flammulina velutipes and Pleurotus eryngii with yields from 130 to 225 µg/g FW. RNA extracted from all samples was intact, as demonstrated by gel electrophoresis and was suitable for downstream molecular applications, including RT-PCR and qPCR.

Chlamydomonas IFT25 is dispensable for flagellar assembly but required to export the BBSome from flagella.

Intraflagellar transport (IFT) particles are composed of polyprotein complexes IFT-A and IFT-B as well as cargo adaptors such as the BBSome. Two IFT-B subunits, IFT25 and IFT27 were found to form a heterodimer, which is essential in exporting the BBSome out of the cilium but not involved in flagellar assembly and cytokinesis in vertebrates. Controversial results were, however, recorded to show that defects in IFT, flagellar assembly and even cytokinesis were caused by IFT27 knockdown in Chlamydomonas reinhardtii Using C. reinhardtii as a model organism, we report that depletion of IFT25 has no effect on flagellar assembly and does not affect the entry of the BBSome into the flagellum, but IFT25 depletion did impair BBSome movement out of the flagellum, clarifying the evolutionally conserved role of IFT25 in regulating the exit of the BBSome from the flagellum cross species. Interestingly, depletion of IFT25 causes dramatic reduction of IFT27 as expected, which does not cause defects in flagellar assembly and cytokinesis in C. reinhardtii Our data thus support that Chlamydomonas IFT27, like its vertebrate homologues, is not involved in flagellar assembly and cytokinesis.