Protein expression and purification最新文献_第2页

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

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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

{"title":"A carboxymethyl cellulase from the yeast Cryptococcus gattii WM276: Expression, purification and characterisation","authors":"","doi":"10.1016/j.pep.2024.106594","DOIUrl":"10.1016/j.pep.2024.106594","url":null,"abstract":"<div><p><em>Cryptococcus gattii</em> 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 <em>C. gattii</em> (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 <em>Escherichia coli</em> 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<sup>+</sup>. 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.</p></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":null,"pages":null},"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}

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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

{"title":"Use of waste frying oil and coconut pulp for the production, isolation, and characterization of a new lipase from Moesziomyces aphidis","authors":"","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":null,"pages":null},"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}

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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

{"title":"Removal and monitoring of residual nucleic acids from core streptavidin inclusion bodies for increased refolding yield","authors":"","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":null,"pages":null},"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

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

{"title":"Partition coefficient screening – An effective approach for finding the best conditions for byproduct removal as demonstrated by a bispecific antibody purification case","authors":"","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":null,"pages":null},"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

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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

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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

{"title":"MAM7 from Vibrio parahaemolyticus: Expression, purification and effects on RAW264.7 cells","authors":"","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":null,"pages":null},"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

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

{"title":"Improved self-cleaving precipitation tags for efficient column free bioseparations","authors":"","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":null,"pages":null},"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