Protein expression and purification最新文献

N-linked glycosylation affects catalytic parameters and fluctuation of the active center of Aspergillus awamori exo-inulinase. N-连接的糖基化影响黑曲霉 awamori 外胰岛素酶活性中心的催化参数和波动。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-30 DOI: 10.1016/j.pep.2024.106613

Anna Dotsenko, Yury Denisenko, Ivan Zorov, Aleksandra Rozhkova, Igor Shashkov

{"title":"N-linked glycosylation affects catalytic parameters and fluctuation of the active center of Aspergillus awamori exo-inulinase.","authors":"Anna Dotsenko, Yury Denisenko, Ivan Zorov, Aleksandra Rozhkova, Igor Shashkov","doi":"10.1016/j.pep.2024.106613","DOIUrl":"10.1016/j.pep.2024.106613","url":null,"abstract":"Heterogeneous expression of enzymes allows large-scale production with reduced costs. Changes in glycosylation often occur due to changes in the expression host. In the study, the catalytic and biochemical properties of Aspergillus awamori exo-inulinase 1 are compared for A. awamori and Penicillium verruculosum expression hosts. The tertiary structure contains seven sites of N-glycosylation, with two of them located near the active center. If expressed in P. verruculosum, the enzyme was four times less glycosylated and two times more active toward sucrose, raffinose, and stachyose due to an increase in kcat. These substrates with a short chain of 2-4 monosaccharide units were used to characterize the interaction of the substrate with the amino acid residues in the active center while preventing the interaction of the substrate with N-linked glycans. Molecular dynamics simulations showed an increase in the fluctuation of the active center with an increase in the length of N-linked glycans. The fluctuation of the residues N40 and Q57, which interact with the hydroxyl group O5 of the fructose unit in the -1 subsite of the active center, was increased by 1.6 times. The fluctuation of the residue W335, which interacts with the hydroxyl group O1 of the fructose unit together with the catalytic residue D41 and affects the torsion angle geometry of the substrate molecules, was increased by 1.5 times. The residue R188, which analogously to W335 affects the torsion angle geometry of the substrate molecules, was also among the affected residues with a 1.2-fold increase in the fluctuation.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142366345","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: characterization of the enzyme's in vitro truncation by thiol endopeptidase activity. 从模式植物拟南芥中异源表达和纯化谷氨酸脱羧酶-1：通过硫醇内肽酶活性鉴定体外截短酶的特性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-27 DOI: 10.1016/j.pep.2024.106612

Brittany S Menard, Kirsten H Benidickson, Lee Marie Raytek, Wayne A Snedden, William C Plaxton

{"title":"Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana: characterization of the enzyme's in vitro truncation by thiol endopeptidase activity.","authors":"Brittany S Menard, Kirsten H Benidickson, Lee Marie Raytek, Wayne A Snedden, William C Plaxton","doi":"10.1016/j.pep.2024.106612","DOIUrl":"https://doi.org/10.1016/j.pep.2024.106612","url":null,"abstract":"Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40% less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes.","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142352672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Evaluation of BVDV E2 proteins based on recombinant baculovirus expression system production as diagnostic antigens and immunogens 基于重组杆状病毒表达系统生产的 BVDV E2 蛋白作为诊断抗原和免疫原的评估

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-22 DOI: 10.1016/j.pep.2024.106611

{"title":"Evaluation of BVDV E2 proteins based on recombinant baculovirus expression system production as diagnostic antigens and immunogens","authors":"","doi":"10.1016/j.pep.2024.106611","DOIUrl":"10.1016/j.pep.2024.106611","url":null,"abstract":"<div><div>Bovine viral diarrhea virus (BVDV) is a significant immunosuppressive pathogen that has a major impact on the global cattle industry. Research efforts are currently focused on the envelope glycoprotein E2 of BVDV to improve immune responses. However, the full-length E2 protein is not ideal as an immune antigen and diagnostic tool, leading to the exploration of alternative strategies. In this study, we optimized the E2 gene using IDEB and ExpOptimizer software, then expressed the E2 gene using both baculovirus and E. coli expression systems. Subsequently, we assessed the immunogenicity of the purified E2 protein in mice and its application in indirect ELISA assays. Our findings showed that the Bac-E2 protein produced by the baculovirus system induced higher levels of antibody production and splenic lymphocyte proliferation in mice compared to the E. coli system. Moreover, the indirect ELISA assay developed using Bac-E2 protein exhibited superior specificity, sensitivity, and accuracy in comparison to the E. coli-expressed E2 ELISA. Overall, our study demonstrates that the optimized E2 protein generated through a baculovirus expression system elicits robust humoral and cellular immune responses in mice, making it a promising candidate for vaccine development. Furthermore, the optimized E2 protein ELISA assay shows enhanced sensitivity and accuracy, suggesting its potential as a valuable diagnostic antigen.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142314941","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii 植物乳杆菌提取的细菌素对耐多药鲍曼不动杆菌的抗生物膜和抗菌作用

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-19 DOI: 10.1016/j.pep.2024.106610

{"title":"Anti-biofilm and antibacterial effect of bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii","authors":"","doi":"10.1016/j.pep.2024.106610","DOIUrl":"10.1016/j.pep.2024.106610","url":null,"abstract":"<div><div>This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 μg/mL, while the MBCs varied between 60 and 120 μg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59 % less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52 % compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142293976","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cloning and characterization of a novel nitric oxide synthase gene from Exiguobacterium profundum and its expression in Escherichia coli 一氧化氮合成酶基因的克隆和特性鉴定及其在大肠杆菌中的表达。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-18 DOI: 10.1016/j.pep.2024.106609

引用次数: 0

Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus 抗副溶血性弧菌的海洋内溶菌素 LysVPB 的特征

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-16 DOI: 10.1016/j.pep.2024.106608

{"title":"Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus","authors":"","doi":"10.1016/j.pep.2024.106608","DOIUrl":"10.1016/j.pep.2024.106608","url":null,"abstract":"<div>Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60 % of its maximum activity observed within the broad pH range of 6.0–10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba2+, Co2+, and Cu2+ showed inhibitory effects on the activity of LysVPB, while Ca2+ can boost it to 126.8 %. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142239835","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops) 对薰衣草扇贝中非特异性脂质转移蛋白 nsLTP 功能机制的认识

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-10 DOI: 10.1016/j.pep.2024.106607

引用次数: 0

Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis 来自 Rahnella aquatilis 的新型 L-isoleucine-4-dioxygenase (RaIDO) 的生物化学和结构特征。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-06 DOI: 10.1016/j.pep.2024.106604

{"title":"Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis","authors":"","doi":"10.1016/j.pep.2024.106604","DOIUrl":"10.1016/j.pep.2024.106604","url":null,"abstract":"<div>The L-isoleucine-4-dioxygenase converts L-isoleucine (Ile) into(2S,3R,4S)-4-(OH)-isoleucine (4-HIL), a naturally occurring hydroxyl amino acid, which is a promising compound for drug and functional food development. Here, a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis was cloned, expressed and characterized, as one of only a few reported L-isoleucine-4-dioxygenases. RaIDO showed high catalytic efficiency with Ile as the substrate, as well as good stability. HPLC-MS and NMR confirmed that RaIDO converts Ile into (2S,3R,4S)-4-(OH)-isoleucine. Further, structural analysis of RaIDO revealed key active site residues, including H159, D161 and H212. The RaIDO enzyme showed an optimal reaction temperature range of 30°C–45 °C, with the highest catalytic activity observed at 40 °C. Additionally, the enzyme exhibited an optimal pH of 8.0. Thus, the novel L-isoleucine-4-dioxygenase (RaIDO) has high catalytic efficiency and good stability, making it a strong candidate for industrial applications.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142146096","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development of an effective method for purifying trypsin using a recombinant inhibitor 开发一种利用重组抑制剂纯化胰蛋白酶的有效方法。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-09-02 DOI: 10.1016/j.pep.2024.106597

{"title":"Development of an effective method for purifying trypsin using a recombinant inhibitor","authors":"","doi":"10.1016/j.pep.2024.106597","DOIUrl":"10.1016/j.pep.2024.106597","url":null,"abstract":"<div>A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30–50 °C and pH range of 4.0–10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-09-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142133522","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells 改善中国仓鼠卵巢细胞（CHO）中重组人绒毛膜促性腺激素表达的策略。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-31 DOI: 10.1016/j.pep.2024.106596

{"title":"Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells","authors":"","doi":"10.1016/j.pep.2024.106596","DOIUrl":"10.1016/j.pep.2024.106596","url":null,"abstract":"<div>Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 μg/ml) and (14.80 ± 0.13 μg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"PeriodicalIF":1.4,"publicationDate":"2024-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142111351","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0