[Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody].

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

[Preparation of recombinant mussel mucin Mfp-3P and its promotion of wound healing].

To investigate the role of recombinant mussel mucin in wound healing, we aimed to prepare this mucin using Pichia pastoris as the host microbe. Our method involved constructing a genetically engineered strain of P. pastoris that expressed a fusion protein consisting of Mfp-3 and preCol-P peptide segments of mussel. After fermentation and purification, we obtained a pure recombinant mussel mucin product. We then conducted experiments to evaluate its effect at both the cellular and animal levels. At the cellular level, we examined its impact on the proliferation and migration of mouse fibroblast L929. At the animal level, we assessed its ability to promote wound healing after full-layer skin resection in rats. Our results showed that the recombinant mussel mucin protein has a content of 90.28% and a purity of 96.49%. The content of 3,4-dihydroxyphenylalanine (DOPA) was 0.73 wt%, and the endotoxin content was less than 0.5 EU/mg. Importantly, the recombinant mussel mucin protein significantly promoted both the migration and proliferation of mouse fibroblast, as well as the wound healing in rat skin. In conclusion, our findings demonstrate that recombinant mussel mucin has the potential to promote wound healing and can be considered a promising medical biomaterial.

[Human tau N-terminal domain-specific monoclonal antibodies: screening and application in blood detection].

The antibodies to the microtubule-associated protein tau play a role in basic and clinical studies of Alzheimer's disease (AD) and other tauopathies. With the recombinant human tau441 as the immunogen, the hybridoma cell strains secreting the anti-human tau N-terminal domain (NTD-tau) monoclonal antibodies were generated by cell fusion and screened by limiting dilution. The purified monoclonal antibodies were obtained by inducing the mouse ascites and affinity chromatography. The sensitivity and specificity of the monoclonal antibodies were examined by indirect ELISA and Western blotting, respectively. A double antibody sandwich ELISA method for detecting human tau protein was established and optimized. The results showed that the positive cloning rate of hybridoma cells was 83.6%. A stable cell line producing ZD8F7 antibodies was established, and the antibody titer in the supernatant of the cell line was 1:16 000. The antibody titer in the ascitic fluid was higher than 1:256 000; and the titer of purified ZD8F7 monoclonal antibodies was higher than 1:128 000. The epitope analysis showed that the ZD8F7 antibody recognized tau21-37 amino acid in the N-terminal domain. The Western blotting results showed that the ZD8F7 antibody recognized the recombinant human tau protein of 50-70 kDa and the human tau protein of 50 kDa in the brain tissue of transgenic AD model mice (APP/PS1/tau). With ZD8F7 as a capture antibody, a quantitative detection method for human tau protein was established, which showed a linear range of 7.8-500.0 pg/mL and could identify human tau protein in the brain tissue of AD transgenic mice and human plasma but not recognize the mouse tau protein. In conclusion, the human NTD-tau-specific monoclonal antibody and the double antibody sandwich ELISA method established in this study are highly sensitive and can serve as a powerful tool for the detection of tau protein in neurodegenerative diseases.

Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis.

Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Exploring class III cellobiose dehydrogenase: sequence analysis and optimized recombinant expression.

BACKGROUND: Cellobiose dehydrogenase (CDH) is an extracellular fungal oxidoreductase with multiple functions in plant biomass degradation. Its primary function as an auxiliary enzyme of lytic polysaccharide monooxygenase (LPMO) facilitates the efficient depolymerization of cellulose, hemicelluloses and other carbohydrate-based polymers. The synergistic action of CDH and LPMO that supports biomass-degrading hydrolases holds significant promise to harness renewable resources for the production of biofuels, chemicals, and modified materials in an environmentally sustainable manner. While previous phylogenetic analyses have identified four distinct classes of CDHs, only class I and II have been biochemically characterized so far. RESULTS: Following a comprehensive database search aimed at identifying CDH sequences belonging to the so far uncharacterized class III for subsequent expression and biochemical characterization, we have curated an extensive compilation of putative CDH amino acid sequences. A sequence similarity network analysis was used to cluster them into the four distinct CDH classes. A total of 1237 sequences encoding putative class III CDHs were extracted from the network and used for phylogenetic analyses. The obtained phylogenetic tree was used to guide the selection of 11 cdhIII genes for recombinant expression in Komagataella phaffii. A small-scale expression screening procedure identified a promising cdhIII gene originating from the plant pathogen Fusarium solani (FsCDH), which was selected for expression optimization by signal peptide shuffling and subsequent production in a 5-L bioreactor. The purified FsCDH exhibits a UV-Vis spectrum and enzymatic activity similar to other characterized CDH classes. CONCLUSION: The successful production and functional characterization of FsCDH proved that class III CDHs are catalytical active enzymes resembling the key properties of class I and class II CDHs. A detailed biochemical characterization based on the established expression and purification strategy can provide new insights into the evolutionary process shaping CDHs and leading to their differentiation into the four distinct classes. The findings have the potential to broaden our understanding of the biocatalytic application of CDH and LPMO for the oxidative depolymerization of polysaccharides.

N-Terminal Amino Acid Affects the Translation Efficiency at Lower Temperatures in a Reconstituted Protein Synthesis System.

The Escherichia coli (E. coli)-based protein synthesis using recombinant elements (PURE) system is a cell-free protein synthesis system reconstituted from purified factors essential for E. coli translation. The PURE system is widely used for basic and synthetic biology applications. One of the major challenges associated with the PURE system is that the protein yield of the system varies depending on the protein. Studies have reported that the efficiency of translation is significantly affected by nucleotide and amino acid sequences, especially in the N-terminal region. Here, we investigated the inherent effect of various N-terminal sequences on protein synthesis using the PURE system. We found that a single amino acid substitution in the N-terminal region significantly altered translation efficiency in the PURE system, especially at low temperatures. This result gives us useful suggestions for the expression of the protein of interest in vitro and in vivo.

An engineered Pichia pastoris platform for the biosynthesis of silk-based nanomaterials with therapeutic potential.

Silk fibroin (SF) from the cocoon of silkworm has exceptional mechanical properties and biocompatibility and is used as a biomaterial in a variety of fields. Sustainable, affordable, and scalable manufacturing of SF would enable its large-scale use. We report for the first time the high-level secretory production of recombinant SF peptides in engineered Pichia pastoris cell factories and the processing thereof to nanomaterials. Two SF peptides (BmSPR3 and BmSPR4) were synthesized and secreted by P. pastoris using signal peptides and appropriate spacing between hydrophilic sequences. By strain engineering to reduce protein degradation, increase glycyl-tRNA supply, and improve protein secretion, we created the optimized P. pastoris chassis PPGSP-8 to produce BmSPR3 and BmSPR4. The SF fed-batch fermentation titers of the resulting two P. pastoris cell factories were 11.39 and 9.48 g/L, respectively. Protein self-assembly was inhibited by adding Tween 80 to the medium. Recombinant SF peptides were processed to nanoparticles (NPs) and nanofibrils. The physicochemical properties of nanoparticles R3NPs and R4NPs from the recombinant SFs synthesized in P. pastoris cell factories were similar or superior to those of RSFNPs (Regenerated Silk Fibroin NanoParticles) originating from commercially available SF. Our work will facilitate the production by microbial fermentation of functional SF for use as a biomaterial.

Expression and characterization of a novel ß-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

An important consideration when expressing mAbs in Escherichiacoli.

Monoclonal antibodies (mAbs) are a driving force in the biopharmaceutical industry. Therapeutic mAbs are usually produced in mammalian cells, but there has been a push towards the use of alternative production hosts, such as Escherichia coli. When the genes encoding for a mAb heavy and light chains are codon-optimized for E. coli expression, a truncated form of the heavy chain can form along with the full-length product. In this work, the role of codon optimization in the formation of a truncated product was investigated. This study used the amino acid sequences of several therapeutic mAbs and multiple optimization algorithms. It was found that several algorithms incorporate sequences that lead to a truncated product. Approaches to avoid this truncated form are discussed.

A new method for the robust expression and single-step purification of dCas9 for CRISPR interference/activation (CRISPRi/a) applications.

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated enzyme 9) is known for its simplicity, versatility, and scalability in genome editing applications. In vitro Cas9, when complexed with sgRNA, binds and cleaves the complementary target sequences with almost perfect precision. The enzyme is exploited for various applications in understanding and changing gene function. dCas9 (deactivated or dead Cas9) is a double mutated version of Cas9 that bears mutations in the nuclease domains of the enzyme and thus cannot cleave the target DNA. dCas9 is equally advantageous since it can alter gene expression using various transcriptional activators CRISPRa and repressors CRISPRi. Additionally, dCas9 can bind to the desired target gene without cleaving it, making it a unique reagent to study the kinetics and stability of RNA-protein-DNA interactions required to design more efficient and specific gene-editing nucleases. An appreciable quantity of pure and homogeneous protein is needed to characterise dCas9 for its structural and functional understanding. This study used an N-terminal acidic tag to express the dCas9 in an E. coli-bacterial host. A simple single-step protocol for robust and efficient production of dCas9 has been described. The study and methods are distinctive as the purification is performed in a single step using inexpensive multi-modal hydroxyapatite chromatography. The purified protein can be used in different in vitro and in vivo studies.

Understanding exopolysaccharide byproduct formation in Komagataella phaffii fermentation processes for recombinant protein production.

BACKGROUND: Komagataella phaffii (Pichia pastoris) has emerged as a common and robust biotechnological platform organism, to produce recombinant proteins and other bioproducts of commercial interest. Key advantage of K. phaffii is the secretion of recombinant proteins, coupled with a low host protein secretion. This facilitates downstream processing, resulting in high purity of the target protein. However, a significant but often overlooked aspect is the presence of an unknown polysaccharide impurity in the supernatant. Surprisingly, this impurity has received limited attention in the literature, and its presence and quantification are rarely addressed. RESULTS: This study aims to quantify this exopolysaccharide in high cell density recombinant protein production processes and identify its origin. In stirred tank fed-batch fermentations with a maximal cell dry weight of 155 g/L, the polysaccharide concentration in the supernatant can reach up to 8.7 g/L. This level is similar to the achievable target protein concentration. Importantly, the results demonstrate that exopolysaccharide production is independent of the substrate and the protein production process itself. Instead, it is directly correlated with biomass formation and proportional to cell dry weight. Cell lysis can confidently be ruled out as the source of this exopolysaccharide in the culture medium. Furthermore, the polysaccharide secretion can be linked to a mutation in the HOC1 gene, featured by all derivatives of strain NRRL Y-11430, leading to a characteristic thinner cell wall. CONCLUSIONS: This research sheds light on a previously disregarded aspect of K. phaffii fermentations, emphasizing the importance of monitoring and addressing the exopolysaccharide impurity in biotechnological applications, independent of the recombinant protein produced.

Expression, purification, and crystal structure of mpox virus A41 protein.

Mpox is a zoonotic disease that was once endemic in Africa countries caused by mpox virus. However, cases recently have been confirmed in many non-endemic countries outside of Africa. The rapidly increasing number of confirmed mpox cases poses a threat to the international community. In-depth studies of key viral factors are urgently needed, which will inform the design of multiple antiviral agents. Mpox virus A41L gene encodes a secreted protein, A41, that is nonessential for viral replication, but could affect the host response to infection via interacting with chemokines. Here, mpox virus A41 protein was expressed in Sf9 cells, and purified by affinity chromatography followed by gel filtration. Surface plasmon resonance spectroscopy showed that purified A41 binds a certain human chemokine CXCL8 with the equilibrium dissociation constant (KD) being 1.22 × 10-6 M. The crystal structure of mpox virus A41 protein was solved at 1.92 Å. Structural analysis and comparison revealed that mpox virus A41 protein adopts a characteristic ß-sheet topology, showing minor differences with that of vaccinia virus. These preliminary structural and functional studies of A41 protein from mpox virus will help us better understand its role in chemokine subversion, and contributing to the knowledge to viral chemokine binding proteins.

Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

High-level production of chitinase by multi-strategy combination optimization in Bacillus licheniformis.

In view of the extensive potential applications of chitinase (ChiA) in various fields such as agriculture, environmental protection, medicine, and biotechnology, the development of a high-yielding strain capable of producing chitinase with enhanced activity holds significant importance. The objective of this study was to utilize the extracellular chitinase from Bacillus thuringiensis as the target, and Bacillus licheniformis as the expression host to achieve heterologous expression of ChiA with enhanced activity. Initially, through structural analysis and molecular dynamics simulation, we identified key amino acids to improve the enzymatic performance of chitinase, and the specific activity of chitinase mutant D116N/E118N was 48% higher than that of the natural enzyme, with concomitant enhancements in thermostability and pH stability. Subsequently, the expression elements of ChiA(D116N/E118N) were screened and modified in Bacillus licheniformis, resulting in extracellular ChiA activity reached 89.31 U/mL. Further efforts involved the successful knockout of extracellular protease genes aprE, bprA and epr, along with the gene clusters involved in the synthesis of by-products such as bacitracin and lichenin from Bacillus licheniformis. This led to the development of a recombinant strain, DW2△abelA, which exhibited a remarkable improvement in chitinase activity, reaching 145.56 U/mL. To further improve chitinase activity, a chitinase expression frame was integrated into the genome of DW2△abelA, resulting in a significant increas to 180.26 U/mL. Optimization of fermentation conditions and medium components further boosted shake flask enzyme activity shake flask enzyme activity, achieving 200.28 U/mL, while scale-up fermentation experiments yielded an impressive enzyme activity of 338.79 U/mL. Through host genetic modification, expression optimization and fermentation optimization, a high-yielding ChiA strain was successfully constructed, which will provide a solid foundation for the extracellular production of ChiA.

Expression, purification and preliminary crystallographic analysis of bacterial transmembrane protein EfeU for iron import.

The bacterial Efe system functions as an importer of free Fe2+ into cells independently of iron-chelating compounds such as siderophores and consisted of iron-binding protein EfeO, peroxidase EfeB, and transmembrane permease EfeU. While we and other researchers reported crystal structures of EfeO and EfeB, that of EfeU remains undetermined. In this study, we constructed expression system of EfeU derived from Escherichia coli, selected E. coli Rosetta-gami 2 (DE3) as an expression host, and succeeded in purification of the proteins which were indicated to form an oligomer by blue native PAGE. We obtained preliminary data of the X-ray crystallography, suggesting that expression and purification methods we established in this study enable structural analysis of the bacterial Efe system.

Expression and biochemical characterization of a novel thermostable alkaline ß-1,3-1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1.

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

The calcium-binding photoprotein clytin II: Expression of the preferred human codon-optimized clytin II gene in Chinese hamster ovary-K1 cells and its use in the G-protein-coupled receptor assays.

Clytin II (CLII) is a Ca2+-binding photoprotein and has been identified as an isotype of clytin I (CLI). CLII consists of apoCLII (an apoprotein) and 2-peroxide of coelenterazine (an adduct of molecular oxygen to coelenterazine), which is identical to the widely used Ca2+-binding photoprotein, aequorin (AQ). However, CLII triggered by Ca2+ exhibits a 4.5-fold higher maximum luminescence intensity (Imax) compared to both AQ and CLI, and it is approximately 5 times less sensitive to Ca2+ than AQ. To confirm the suitability of the preferred human codon-optimized CLII (pCLII) gene for cell-based G-protein-coupled receptor (GPCR) assays, a transformant stably expressing apoprotein of pCLII using the pCLII gene in the mitochondria of CHO-K1 cells was established and in situ regenerated pCLII in the cells were applied to the high-throughput screening system. An ATP-stimulated GPCR assay for endogenous P2Y purinergic receptors was confirmed using the established stable transformant.

[Identification and expression pattern analysis of a secretory phospholipase A2 gene in Bombyx mori].

Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori (BmsPLA2-1-1). In this study, we further identified four new sPLA2 genes (BmsPLA2-1-2, BmsPLA2-2, BmsPLA2-3, and BmsPLA2-4) in B. mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B. mori. The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus, the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.