Protein expression and purification最新文献_第8页

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

Dafeng Liu , Wenrui Dou , Hongying Song , Huashui Deng , Zhu Tian , Rong Chen , Zhen Liu , Ziwei Jiao , Oren Akhberdi

{"title":"Insights into the functional mechanism of the non-specific lipid transfer protein nsLTP in Kalanchoe fedtschenkoi (Lavender scallops)","authors":"Dafeng Liu , Wenrui Dou , Hongying Song , Huashui Deng , Zhu Tian , Rong Chen , Zhen Liu , Ziwei Jiao , Oren Akhberdi","doi":"10.1016/j.pep.2024.106607","DOIUrl":"10.1016/j.pep.2024.106607","url":null,"abstract":"<div>Plant non-specific lipid transfer protein (nsLTP) is able to bind and transport lipids and essential oils, as well as engage in various physiological processes, including defense against phytopathogens. Kalanchoe fedtschenkoi (Lavender Scallops) is an attractive and versatile succulent. To investigate the functional mechanism of Kalanchoe fedtschenkoi nsLTP (Ka-nsLTP), we expressed, purified and successfully obtained monomeric Ka-nsLTP. Mutational experiments revealed that the C6A variant retained the same activity as the wild-type (WT) Ka-nsLTP. Ka-nsLTP showed weak antiphytopathogenic bacterial activity, but inhibited fungal growth. Ka-nsLTP possessed a hydrophobic cavity effectively binding lauric acid. Our results offer novel molecular insights into the functional mechanism of nsLTP, which broadens our knowledge of the biological function of nsLTP in crops and provides a useful locus for genetic improvement of plants.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106607"},"PeriodicalIF":1.4,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142164833","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

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

Ruida Shan , Yishu Wang , Shuxin Cheng , Xia Li , Xiaohui Yang , Dengyue Sun , Piwu Li

{"title":"Biochemical and structural characterization of a novel L-isoleucine-4-dioxygenase (RaIDO) from Rahnella aquatilis","authors":"Ruida Shan , Yishu Wang , Shuxin Cheng , Xia Li , Xiaohui Yang , Dengyue Sun , Piwu Li","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":"226 ","pages":"Article 106604"},"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

Chen Li , Zhaoxia Wang , Zejie Niu , Jiao Li , Lanxin Chen , Xiaodong Cui , Fang Li

{"title":"Development of an effective method for purifying trypsin using a recombinant inhibitor","authors":"Chen Li , Zhaoxia Wang , Zejie Niu , Jiao Li , Lanxin Chen , Xiaodong Cui , Fang Li","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":"225 ","pages":"Article 106597"},"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

Iheb Boukari , Samia Rourou , Dorsaf Bouzazi , Khadija Essafi-Benkhadir , Héla Kallel

{"title":"Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells","authors":"Iheb Boukari , Samia Rourou , Dorsaf Bouzazi , Khadija Essafi-Benkhadir , Héla Kallel","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":"225 ","pages":"Article 106596"},"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

A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium 从杂交瘤培养基中制备生物素化免疫球蛋白 M 的简单程序。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-26 DOI: 10.1016/j.pep.2024.106595

Tetsuya Okuda , Katsuya Kato

引用次数: 0

A carboxymethyl cellulase from the yeast Cryptococcus gattii WM276: Expression, purification and characterisation 一种来自隐球菌 WM276 的羧甲基纤维素酶：表达、纯化和表征

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-26 DOI: 10.1016/j.pep.2024.106594

Dylan Moodley, Angela Botes

{"title":"A carboxymethyl cellulase from the yeast Cryptococcus gattii WM276: Expression, purification and characterisation","authors":"Dylan Moodley, Angela Botes","doi":"10.1016/j.pep.2024.106594","DOIUrl":"10.1016/j.pep.2024.106594","url":null,"abstract":"<div>Cryptococcus gattii and its medical implications have been extensively studied. There is, however, a significant knowledge gap regarding cryptococcal survival in its environmental niche, namely woody material, which is glaring given that infection is linked to environmental populations. A gene from C. gattii (WM276), the predominant global molecular type (VGI), has been sequenced and annotated as a putative cellulase. It is therefore, of both medical and industrial intertest to delineate the structure and function of this enzyme. A homology model of the enzyme was constructed as a fusion protein to a maltose binding protein (MBP). The CGB_E4160W gene was overexpressed as an MBP fusion enzyme in Escherichia coli T7 cells and purified to homogeneity using amylose affinity chromatography. The structural and functional character of the enzyme was investigated using fluorescence spectroscopy and enzyme activity assays, respectively. The optimal enzyme pH and temperature were found to be 6.0 and 50 °C, respectively, with an optimal salt concentration of 500 mM. Secondary structure analysis using Far-UV CD reveals that the MBP fusion protein is primarily α-helical with some β-sheets. Intrinsic tryptophan fluorescence illustrates that the MBP-cellulase undergoes a conformational change in the presence of its substrate, CMC-Na+. The thermotolerant and halotolerant nature of this particular cellulase, makes it useful for industrial applications, and adds to our understanding of the pathogen's environmental physiology.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106594"},"PeriodicalIF":1.4,"publicationDate":"2024-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824001669/pdfft?md5=0ce3a90264b1d65ac8b9e512dd0583ef&pid=1-s2.0-S1046592824001669-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142088967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Use of waste frying oil and coconut pulp for the production, isolation, and characterization of a new lipase from Moesziomyces aphidis 利用废弃煎炸油和椰子浆生产、分离和鉴定蚜茧霉菌的新型脂肪酶。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-22 DOI: 10.1016/j.pep.2024.106584

Alexsandra Nascimento Ferreira , Tatielle Pereira Silva , Ciro Ramon Félix , Julia Lins Lopes , Cláudio Wiliam Victor dos Santos , Dávida Maria Ribeiro Cardoso dos Santos , Melissa Fontes Landell , Francis Soares Gomes , Hugo Juarez Vieira Pereira

{"title":"Use of waste frying oil and coconut pulp for the production, isolation, and characterization of a new lipase from Moesziomyces aphidis","authors":"Alexsandra Nascimento Ferreira , Tatielle Pereira Silva , Ciro Ramon Félix , Julia Lins Lopes , Cláudio Wiliam Victor dos Santos , Dávida Maria Ribeiro Cardoso dos Santos , Melissa Fontes Landell , Francis Soares Gomes , Hugo Juarez Vieira Pereira","doi":"10.1016/j.pep.2024.106584","DOIUrl":"10.1016/j.pep.2024.106584","url":null,"abstract":"<div>Lipases comprise the third most commercialized group of enzymes worldwide and those of microbial origin are sought for their multiple advantages. Agro-industrial waste can be an alternative culture medium for producing lipases, reducing production costs and the improper disposal of waste frying oil (WFO). This study aimed to produce yeast lipases through submerged fermentation (SF) using domestic edible oil waste as inducer and alternative culture medium. The optimal culture conditions, most effective inducer, and purification method for a new lipase from Moesziomyces aphidis BRT57 were identified. Yeast was cultured in medium containing green coconut pulp and WFO waste for 72 h. The maximum production of lipases in SF occurred in a culture medium containing WFO and yeast extract at 48 and 72 h of incubation, with enzyme activities of 8.88 and 11.39 U mL−1, respectively. The lipase was isolated through ultrafiltration followed by size exclusion chromatography, achieving a 50.46 % recovery rate. To the best of our knowledge, this is the first study to report the production and purification of lipases from M. aphidis, demonstrating the value of frying oil as inducer and alternative medium for SF, contributing to the production of fatty acids for biodiesel from food waste.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106584"},"PeriodicalIF":1.4,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142047101","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

Removal and monitoring of residual nucleic acids from core streptavidin inclusion bodies for increased refolding yield 去除和监测核心链霉亲和素包涵体中的残余核酸，以提高重折叠产量。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-22 DOI: 10.1016/j.pep.2024.106591

Nurul Nadia Mohamad Alias, Eugene Boon Beng Ong, Mervyn W.O. Liew

{"title":"Removal and monitoring of residual nucleic acids from core streptavidin inclusion bodies for increased refolding yield","authors":"Nurul Nadia Mohamad Alias, Eugene Boon Beng Ong, Mervyn W.O. Liew","doi":"10.1016/j.pep.2024.106591","DOIUrl":"10.1016/j.pep.2024.106591","url":null,"abstract":"<div>Commercial production of recombinant streptavidin (SAV) using soluble expression route is cost-prohibitive, resulting from its inherent toxicity toward commercially available Escherichia coli hosts (such as BL21) and low productivity of existing manufacturing processes. Quality challenges can also result from binding of streptavidin in the host cells. One way to overcome these challenges is to allow formation of inclusion bodies (IBs). Nevertheless, carried-over cellular contaminants during IBs preparation can hinder protein refolding and application of SAV in nucleic acid-based applications. Hence, removing associated contaminants in recombinant IBs is imperative for maximum product outcomes. In this study, the IBs isolation method from our group was improved to remove residual DNA found in refolded core SAV (cSAV). The improvements were attained by incorporating quantitative real-time polymerase chain reactions (qPCR) for residual DNA monitoring. We attained 99 % cellular DNA removal from cSAV IBs via additional wash and sonication steps, and the addition of benzonase nuclease during lysis. A 10 % increment of cSAV refolding yield (72 %) and 83 % reduction of residual DNA from refolding of 1 mg cSAV IBs were observed under extensive sonication. Refolding of cSAV was not affected and its activity was not compromised. The optimized process reported here highlights the importance of obtaining cSAV IBs with minimal contaminants prior to refolding to increase product yield, and the usefulness of the qPCR method to monitor nucleic acid removed from each step of the process.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106591"},"PeriodicalIF":1.4,"publicationDate":"2024-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142056328","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

Improved production of class I phosphatidylinositol 4,5-bisphosphate 3-kinase 改进 I 类磷脂酰肌醇-4,5-二磷酸 3-激酶的生产。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-20 DOI: 10.1016/j.pep.2024.106582

Simon Messing, Stephanie R.T. Widmeyer, John-Paul Denson, Jennifer Mehalko, Vanessa E. Wall, Matthew Drew, Kelly Snead, Min Hong, Carissa Grose, Dominic Esposito, William Gillette

引用次数: 0

Partition coefficient screening – An effective approach for finding the best conditions for byproduct removal as demonstrated by a bispecific antibody purification case 分配系数筛选--这是一种有效的方法，可通过双特异性抗体纯化案例找到去除副产品的最佳条件。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-20 DOI: 10.1016/j.pep.2024.106583

Wei Chen , Ting Zhang , Peter K. Wang , Chien-Chun Liao , Yifeng Li , Yan Wan

{"title":"Partition coefficient screening – An effective approach for finding the best conditions for byproduct removal as demonstrated by a bispecific antibody purification case","authors":"Wei Chen , Ting Zhang , Peter K. Wang , Chien-Chun Liao , Yifeng Li , Yan Wan","doi":"10.1016/j.pep.2024.106583","DOIUrl":"10.1016/j.pep.2024.106583","url":null,"abstract":"<div>In recombinant protein purification, differences in isoelectric point (pI)/surface charge and hydrophobicity between the product and byproducts generally form the basis for separation. For bispecific antibodies (bsAbs), in many cases the physicochemical difference between product and byproducts is subtle, making byproduct removal considerably challenging. In a previous report, with a bsAb case study, we showed that partition coefficient (Kp) screening for the product and byproducts under various conditions facilitated finding conditions under which effective separation of two difficult-to-remove byproducts was achieved by anion exchange (AEX) chromatography. In the current work, as a follow-up study, we demonstrated that the same approach enabled identification of conditions allowing equally good byproduct removal by mixed-mode chromatography with remarkably improved yield. Results from the current and previous studies proved that separation factor determination based on Kp screening for product and byproduct is an effective approach for finding conditions enabling efficient and maximum byproduct removal, especially in challenging cases.</div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"225 ","pages":"Article 106583"},"PeriodicalIF":1.4,"publicationDate":"2024-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142018399","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