Protein expression and purification最新文献_第10页

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><p>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 <em>Moesziomyces aphidis</em> 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<sup>−1</sup>, 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 <em>M. aphidis</em>, 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.</p></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><p>Commercial production of recombinant streptavidin (SAV) using soluble expression route is cost-prohibitive, resulting from its inherent toxicity toward commercially available <em>Escherichia coli</em> 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.</p></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><p>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 (K<sub>p</sub>) 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 K<sub>p</sub> screening for product and byproduct is an effective approach for finding conditions enabling efficient and maximum byproduct removal, especially in challenging cases.</p></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

Removal of signal peptide variants by cation exchange chromatography: A case study 阳离子交换色谱法去除信号肽变体：案例研究。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-19 DOI: 10.1016/j.pep.2024.106581

Yong Fu , Yangguang Xu , Maodan Zhang, Fengjuan Lv

引用次数: 0

A multidomain PARP14 construct suitable for bacterial expression 适合细菌表达的多域 PARP14 构建物。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-17 DOI: 10.1016/j.pep.2024.106580

Constantinos Chatzicharalampous, Herwig Schüler

{"title":"A multidomain PARP14 construct suitable for bacterial expression","authors":"Constantinos Chatzicharalampous, Herwig Schüler","doi":"10.1016/j.pep.2024.106580","DOIUrl":"10.1016/j.pep.2024.106580","url":null,"abstract":"<div><p>Poly-ADP-ribose polymerase-14 (PARP14) can modify proteins and nucleic acids by the reversible addition of a single ADP-ribose molecule. Aberrant PARP14 functions have been related to cancer and inflammation, and its domains are involved in processes related to viral infection. Previous research indicates that PARP14 functions might be mediated via a multitude of target proteins. <em>In vitro</em> studies of this large multidomain enzyme have been complicated by difficulties to obtain biochemical quantities of pure protein. Here we present a strategy that allows bacterial expression and purification of a functional multidomain construct of PARP14. We substituted an internal KH domain and its neighboring unstructured region with a SUMO domain to obtain a protein construct that encompasses three macrodomains, a WWE domain, and a PARP catalytic domain. We show that the resulting construct retains both ADP-ribosyltransferase and de-MARylase activities. This construct will be useful in structural and functional studies of PARP14.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106580"},"PeriodicalIF":1.4,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1046592824001529/pdfft?md5=f6c9e5d14a9f3a631e3292300471b274&pid=1-s2.0-S1046592824001529-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142000597","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

Improved self-cleaving precipitation tags for efficient column free bioseparations 用于高效无柱生物分离的改进型自裂解沉淀标记。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-15 DOI: 10.1016/j.pep.2024.106578

Hongyu Yuan , Sai Vivek Prabhala , Michael J. Coolbaugh, Samuel D. Stimple, David W. Wood

{"title":"Improved self-cleaving precipitation tags for efficient column free bioseparations","authors":"Hongyu Yuan , Sai Vivek Prabhala , Michael J. Coolbaugh, Samuel D. Stimple, David W. Wood","doi":"10.1016/j.pep.2024.106578","DOIUrl":"10.1016/j.pep.2024.106578","url":null,"abstract":"<div><p>Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the <em>Mtu RecA</em> ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original <em>Mtu RecA</em> ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106578"},"PeriodicalIF":1.4,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996282","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

MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells 副溶血性弧菌中的 MAM7：表达、纯化及对 RAW264.7 细胞的影响。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-15 DOI: 10.1016/j.pep.2024.106579

Qingsong Zeng, Peifang Lai, Mingqin Huang, Ximing Peng, Junjie Huang, Qintao Chen, Yanxu Chen, Huaqian Wang

{"title":"MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells","authors":"Qingsong Zeng, Peifang Lai, Mingqin Huang, Ximing Peng, Junjie Huang, Qintao Chen, Yanxu Chen, Huaqian Wang","doi":"10.1016/j.pep.2024.106579","DOIUrl":"10.1016/j.pep.2024.106579","url":null,"abstract":"<div><p>V. parahaemolyticus is a Gram-negative bacterium that causes gastroenteritis. Within the realm of bacterial interactions with the gut, the outer membrane protein MAM7 plays a key role. However, the precise function of MAM7 in intestinal inflammation, particularly its interactions with macrophages, remains unclear. In this study, we successfully expressed and purified recombinant MAM7. After optimization of the MAM7 expression condition, it was found that the optimal concentration and temperature were 0.75 mM and 15 °C, respectively, resulting in a 27-fold increase in its yield. Furthermore, RAW264.7 cytotoxicity assay was conducted. The CCK-8 results revealed that MAM7 substantially stimulated the proliferation of RAW264.7 cells, with its optimal concentration determined to be 7.5 μg/mL. Following this, the NO concentration of MAM7 was tested, revealing a significant increase (p < 0.05) in NO levels. Additionally, the relative mRNA levels of IL-1β, IL-6, and TNF-α in RAW264.7 cells were measured by qRT-PCR, showing a remarkable elevation (p < 0.05). Moreover, ELISA results demonstrated that MAM7 effectively stimulated the secretion of IL-6 and TNF-α by RAW264.7 cells. In summary, these findings strongly suggest that MAM7 serves as a proinflammatory adhesion factor with the capacity to modulate immune responses.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106579"},"PeriodicalIF":1.4,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996283","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

Novel heterologously expressed protein, AjPSPLP-3, derived from Apostichopus japonicus exhibits cell proliferation and migration activities 异源表达的新蛋白 AjPSPLP-3 来自日本狎鸥鱼，具有细胞增殖和迁移活性。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-15 DOI: 10.1016/j.pep.2024.106577

Weitao Wang , Yiwei Meng , Xin Yin , Peipei Zhao , Mengmeng Wang , Jingli Ren , Jiyuan Zhang , Lixin Zhang , Yunqian Cui , Xuekui Xia

{"title":"Novel heterologously expressed protein, AjPSPLP-3, derived from Apostichopus japonicus exhibits cell proliferation and migration activities","authors":"Weitao Wang , Yiwei Meng , Xin Yin , Peipei Zhao , Mengmeng Wang , Jingli Ren , Jiyuan Zhang , Lixin Zhang , Yunqian Cui , Xuekui Xia","doi":"10.1016/j.pep.2024.106577","DOIUrl":"10.1016/j.pep.2024.106577","url":null,"abstract":"<div><p>Developing more effective bioactive ingredients of natural origin is imperative for promoting wound healing. Sea cucumbers have long enjoyed a good reputation as both food delicacies and traditional medicines. In this study, we heterogeneously expressed a <em>Apostichopus japonicus</em> derived novel protein AjPSPLP-3, which exhibits a theoretical molecular weight of 13.034 kDa, through fusion with maltose binding protein (MBP). AjPSPLP-3 contains a strict CXXCXC motif, nine extremely conserved cysteine residues and two highly conserved cysteine residues. The predicted structure of AjPSPLP-3 consists of random coil and nine β-sheets, Cys<sup>30</sup>-Cys<sup>67</sup>, Cys<sup>38</sup>-Cys<sup>58</sup>, Cys<sup>53</sup>-Cys<sup>90</sup>, Cys<sup>56</sup>-Cys<sup>66</sup>, and Cys<sup>81</sup>-Cys<sup>102</sup> participating in the formation of five pairs of disulfide bonds. <em>In vitro</em> experiments conducted on HaCaT cells proved that AjPSPLP-3 and MBP-fused AjPSPLP-3 significantly contribute to HaCaT cells proliferation and migration without exhibiting hemolytic activity on murine erythrocytes. Specifically, treatment with 10 μmol/L MBP-fused AjPSPLP-3 protein increased the viability of HaCaT cells by 12.28 % (p < 0.001), while treatment with 10 μmol/L AjPSPLP-3 protein increased viability of HaCaT cells by 6.01 % (p < 0.01). Furthermore, wound closure of MBP-fused AjPSPLP-3 and AjPSPLP-3 were 22.51 % (p < 0.01) and 7.32 % (p < 0.05) higher than that of the control groups in HaCaT cells following 24 h of incubation.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"224 ","pages":"Article 106577"},"PeriodicalIF":1.4,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141996284","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

Protein inclusion into ice can dissociate subunits 蛋白质加入冰中会使亚单位解离。

IF 1.4 4区生物学

Protein expression and purification Pub Date : 2024-08-11 DOI: 10.1016/j.pep.2024.106576

Robert Eves, Peter L. Davies

引用次数: 0