Pub Date : 2024-11-20DOI: 10.1016/j.pep.2024.106633
Katherine A. Edmonds, Karla Diaz-Rodriguez, David P. Giedroc
The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in Streptococcus pneumoniae, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed S. pneumoniae EgtU and purified it from E. coli BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization.
细菌 ATP 结合盒（ABC）转运体 EgtU 负责摄取肺炎链球菌细胞中的抗氧化剂麦角硫因，它在多种微生物病原体中都有同源物，其种类之多令人惊讶。EgtU 的溶质结合结构域的晶体结构已有报道，但完整的异构四聚体转运体的结构和功能的许多细节仍有待阐明。在本研究中，我们表达了肺炎双球菌的 EgtU，并从大肠杆菌 BL21 (DE3) 中纯化出了高纯度和高均匀度的 EgtU。我们的初步数据证实了在 DDM 胶束中溶解的全长转运体与麦角硫因的结合和 ATP 的水解。我们的工作流程可以分离出适当数量的 EgtU，用于正在进行的结构研究和详细的生物物理表征。
{"title":"Expression and purification of the intact bacterial ergothioneine transporter EgtU","authors":"Katherine A. Edmonds, Karla Diaz-Rodriguez, David P. Giedroc","doi":"10.1016/j.pep.2024.106633","DOIUrl":"10.1016/j.pep.2024.106633","url":null,"abstract":"<div><div>The bacterial ATP-binding cassette (ABC) transporter EgtU is responsible for uptake of the cellular antioxidant ergothioneine in <em>Streptococcus pneumoniae</em>, and it has homologs in a surprisingly diverse range of microbial pathogens. Crystal structures have been reported for the solute binding domain of EgtU, but many details of the structure and function of the intact heterotetrameric transporter remain to be elucidated. In this study, we have expressed <em>S. pneumoniae</em> EgtU and purified it from <em>E. coli</em> BL21 (DE3) with high purity and homogeneity. Our preliminary data establish ergothioneine binding and ATP hydrolysis by the full-length transporter solubilized in DDM micelles. Our workflow allows for isolation of suitable quantities of EgtU for ongoing structural studies and detailed biophysical characterization.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106633"},"PeriodicalIF":1.4,"publicationDate":"2024-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142693469","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}
Pub Date : 2024-11-15DOI: 10.1016/j.pep.2024.106622
Shashank Arora, Pawan Nagarkar, Jacinta S. D'Souza
Forkhead box protein J1 (FOXJ1) is the key transcriptional regulator during the conversion of mammalian primary cilium with a 9 + 0 architecture to the motile (9 + 2) one. The nucleotide sequences of the full-length and DNA-binding domain (DBD) of the open reading frame (ORF) were isolated and expressed into E. coli as 6xHis-tagged proteins. Upon induction, the DBD formed inclusion bodies that solubilized with 8 M urea. No induction of 6xHis-FOXJ1 protein was seen despite sub-cloning into several expression vectors and E. coli host strains. To improve induction and solubility, the 6xHis tag was substituted with Glutathione S-transferase (GST), and weak induction was seen in E. coli BL21(DE3). The GST-FOXJ1 showed anomalous migration on denaturing gel electrophoresis (AM-DRE), migrating at approximately 83 kDa instead of its calculated molecular weight (Mr) of 72.4 kDa. It was also unstable and led to degradation products. The 6xHis tag was substituted with Glutathione S-transferase (GST) to improve induction and solubility. Codon-optimization improved the induction, but the protein still showed AM-DRE and instability. It seemed that the recombinant protein was either toxic or posed a metabolic burden to the E. coli cells or, once produced was prone to degradation due mainly to the lack of post-translational modification (PTM). This process is required for some eukaryotic proteins after they are manufactured in the ribosomal factory. Both the purified recombinant proteins exhibited cysteine-induced oligomerization via the formation of disulphide bridges since this was reduced using dithiothreitol (DTT). Both were equally functional as these individually bound to an oligonucleotide, a consensus DNA-binding sequence for FOX proteins. Further, the recombinant polypeptides corresponding to the C-terminus and N-terminus show anomalies indicating that the highly acidic residues (known as polyacidic gel-shifting domains) in these polypeptides contribute to the AM-DRE. We demonstrate for the first time that the recombinant HsFOXJ1 and its DBD bind to DNA, its polyacidic gel-shifting domains are the reason for the AM-DRE, is unstable leading to degradation products, exhibits cysteine-induced oligomerization and harbours intrinsically disordered regions.
叉头盒蛋白 J1（FOXJ1）是哺乳动物初级纤毛从 9+0 结构向运动（9+2）结构转化过程中的关键转录调节因子。我们分离了开放阅读框（ORF）的全长和DNA结合域（DBD）的核苷酸序列，并将其表达到大肠杆菌中，成为6xHis标记的蛋白质。诱导后，DBD 形成包涵体，并用 8 M 尿素溶解。尽管将 6xHis-FOXJ1 蛋白亚克隆到多种表达载体和大肠杆菌宿主菌株中，但仍未发现 6xHis-FOXJ1 蛋白被诱导。为了提高诱导性和可溶性，用谷胱甘肽 S-转移酶（GST）取代了 6xHis 标记，在大肠杆菌 BL21(DE3) 中发现了微弱的诱导作用。GST-FOXJ1 在变性凝胶电泳（AM-DRE）中显示出异常迁移，迁移分子量约为 83 kDa，而不是其计算分子量（Mr）72.4 kDa。它还不稳定，会产生降解产物。用谷胱甘肽 S-转移酶（GST）取代 6xHis 标记以提高诱导性和溶解性，代码优化提高了诱导性，但蛋白质仍显示 AM-DRE，且不稳定。重组蛋白似乎对大肠杆菌细胞有毒性或造成代谢负担，或者一旦产生就容易降解，主要原因是缺乏翻译后修饰（PTM）。一些真核蛋白质在核糖体工厂制造后需要进行这一过程。两种纯化的重组蛋白都表现出半胱氨酸通过形成二硫键诱导的寡聚化，因为使用二硫苏糖醇（DTT）可将其还原。这两种蛋白的功能相同，都能单独与寡核苷酸结合，而寡核苷酸是 FOX 蛋白的一种共识 DNA 结合序列。此外，与 C 端和 N 端相对应的重组多肽显示出异常，表明这些多肽中的高酸性残基（称为多酸性凝胶转移结构域）有助于 AM-DRE。我们首次证明，重组的 HsFOXJ1 及其 DBD 可与 DNA 结合，其多酸性凝胶移动结构域是产生 AM-DRE 的原因，它不稳定，会产生降解产物，表现出半胱氨酸诱导的寡聚化，并含有内在无序区。
{"title":"Recombinant human FOXJ1 protein binds DNA, forms higher-order oligomers, has gel-shifting domains and contains intrinsically disordered regions","authors":"Shashank Arora, Pawan Nagarkar, Jacinta S. D'Souza","doi":"10.1016/j.pep.2024.106622","DOIUrl":"10.1016/j.pep.2024.106622","url":null,"abstract":"<div><div>Forkhead box protein J1 (FOXJ1) is the key transcriptional regulator during the conversion of mammalian primary cilium with a 9 + 0 architecture to the motile (9 + 2) one. The nucleotide sequences of the full-length and DNA-binding domain (DBD) of the open reading frame (ORF) were isolated and expressed into <em>E. coli</em> as 6xHis-tagged proteins. Upon induction, the DBD formed inclusion bodies that solubilized with 8 M urea. No induction of 6xHis-FOXJ1 protein was seen despite sub-cloning into several expression vectors and <em>E. coli</em> host strains. To improve induction and solubility, the 6xHis tag was substituted with Glutathione S-transferase (GST), and weak induction was seen in <em>E. coli</em> BL21(DE3). The GST-FOXJ1 showed anomalous migration on denaturing gel electrophoresis (AM-DRE), migrating at approximately 83 kDa instead of its calculated molecular weight (<em>Mr</em>) of 72.4 kDa. It was also unstable and led to degradation products. The 6xHis tag was substituted with Glutathione S-transferase (GST) to improve induction and solubility. Codon-optimization improved the induction, but the protein still showed AM-DRE and instability. It seemed that the recombinant protein was either toxic or posed a metabolic burden to the <em>E. coli</em> cells or, once produced was prone to degradation due mainly to the lack of post-translational modification (PTM). This process is required for some eukaryotic proteins after they are manufactured in the ribosomal factory. Both the purified recombinant proteins exhibited cysteine-induced oligomerization <em>via</em> the formation of disulphide bridges since this was reduced using dithiothreitol (DTT). Both were equally functional as these individually bound to an oligonucleotide, a consensus DNA-binding sequence for FOX proteins. Further, the recombinant polypeptides corresponding to the C-terminus and N-terminus show anomalies indicating that the highly acidic residues (known as polyacidic gel-shifting domains) in these polypeptides contribute to the AM-DRE. We demonstrate for the first time that the recombinant HsFOXJ1 and its DBD bind to DNA, its polyacidic gel-shifting domains are the reason for the AM-DRE, is unstable leading to degradation products, exhibits cysteine-induced oligomerization and harbours intrinsically disordered regions.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106622"},"PeriodicalIF":1.4,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142644500","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}
Pub Date : 2024-11-12DOI: 10.1016/j.pep.2024.106623
Aleksandra Chorążewska, Darragh Regan, Marta Kalka, Krzysztof Ciura, Natalia Porębska, Łukasz Opaliński
Numerous proteins in nature strictly require oligomerization for their full activity. Moreover, the function of natural and artificial proteins can me adjusted by altering their oligomeric state, leading to development of biotechnologically-relevant biomacromolecules. Oligomerization scaffolds from natural sources and designed de novo enable shuffling the oligomeric state and valency of biomacromolecules. In this report we probed the scaffolding potential of the thermostable phenylacetic acid degradation protein acyl-CoA from Thermus thermophilus (TtPaaI). We designed and successfully produced the fusion protein between TtPaaI (scaffold) and galectin-7, a multifunctional lectin implicated in human diseases (ligand) and demonstrated that TtPaaI can serve as a framework for functional multivalent display of ligands.
{"title":"Thermostable phenylacetic acid degradation protein TtPaaI from Thermus thermophilus as a scaffold for tetravalent display of proteins","authors":"Aleksandra Chorążewska, Darragh Regan, Marta Kalka, Krzysztof Ciura, Natalia Porębska, Łukasz Opaliński","doi":"10.1016/j.pep.2024.106623","DOIUrl":"10.1016/j.pep.2024.106623","url":null,"abstract":"<div><div>Numerous proteins in nature strictly require oligomerization for their full activity. Moreover, the function of natural and artificial proteins can me adjusted by altering their oligomeric state, leading to development of biotechnologically-relevant biomacromolecules. Oligomerization scaffolds from natural sources and designed <em>de novo</em> enable shuffling the oligomeric state and valency of biomacromolecules. In this report we probed the scaffolding potential of the thermostable phenylacetic acid degradation protein acyl-CoA from <em>Thermus thermophilus</em> (<em>Tt</em>PaaI). We designed and successfully produced the fusion protein between <em>Tt</em>PaaI (scaffold) and galectin-7, a multifunctional lectin implicated in human diseases (ligand) and demonstrated that <em>Tt</em>PaaI can serve as a framework for functional multivalent display of ligands.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106623"},"PeriodicalIF":1.4,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142626845","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}
Pub Date : 2024-11-09DOI: 10.1016/j.pep.2024.106621
Wanli Guo , Dajin Wang , Wei Chen , Chuyang Rao , Yunxuan Tang , Wangfeng Li
Thermostable apurinic/apyrimidinic (AP) endonuclease (TtAP), cloned from Caldanaerobacter subterraneus subsp. tengcongensis, is an exonuclease III (Exo III) family protein with high-heat resistance, has activities of AP site endonuclease, 3′–5′ exonuclease, and 3′-nuclease, and facilitates efficient amplification of lengthy DNA fragments in PCR. However, the research of the combinant TtAP in Escherichia coli with its expression, large-scale extraction and purification of its protein was limited. In this study, we optimized the codons of TtAP gene for expression in E. coli and constructed a fusion gene encoding TtAP with a 6His tag (TtAP-6His). TtAP-6His was put into vector pET-30a(+) to form the expression vector pET-30a(+)-TtAP-6His, and was then introduced into E. coli strain Rosetta (DE3). We established a systematic process for the extraction of TtAP protein using 5 liters of bacterial suspension, including the optimization of IPTG induction time (6 h), followed by protein extraction using enzymolysis buffers, the heat treatment of temperature (70 °C) with 60 min to remove impurity, precipitation with ammonium sulfate (55 %), protein purification with Ni-affinity chromatography, and the enzyme activities finally were determined. The purification yield of TtAP-6His ranged from 73.67 to 115.25 mg/L (47 KU/mg).
{"title":"The heterogeneous expression, extraction, and purification of recombinant Caldanaerobacter subterraneus subsp. tengcongensis apurine/apyrimidine endonuclease in Escherichia coli","authors":"Wanli Guo , Dajin Wang , Wei Chen , Chuyang Rao , Yunxuan Tang , Wangfeng Li","doi":"10.1016/j.pep.2024.106621","DOIUrl":"10.1016/j.pep.2024.106621","url":null,"abstract":"<div><div>Thermostable apurinic/apyrimidinic (AP) endonuclease (TtAP), cloned from <em>Caldanaerobacter subterraneus</em> subsp. tengcongensis, is an exonuclease III (Exo III) family protein with high-heat resistance, has activities of AP site endonuclease, 3′–5′ exonuclease, and 3′-nuclease, and facilitates efficient amplification of lengthy DNA fragments in PCR. However, the research of the combinant <em>TtAP</em> in <em>Escherichia coli</em> with its expression, large-scale extraction and purification of its protein was limited. In this study, we optimized the codons of TtAP gene for expression in <em>E. coli</em> and constructed a fusion gene encoding TtAP with a 6His tag (<em>TtAP-6His</em>). <em>TtAP-6His</em> was put into vector <em>pET-30a</em><sup>(+)</sup> to form the expression vector <em>pET-30a</em><sup>(+)</sup><em>-TtAP-6His</em>, and was then introduced into <em>E. coli</em> strain Rosetta (DE3). We established a systematic process for the extraction of TtAP protein using 5 liters of bacterial suspension, including the optimization of IPTG induction time (6 h), followed by protein extraction using enzymolysis buffers, the heat treatment of temperature (70 °C) with 60 min to remove impurity, precipitation with ammonium sulfate (55 %), protein purification with Ni-affinity chromatography, and the enzyme activities finally were determined. The purification yield of TtAP-6His ranged from 73.67 to 115.25 mg/L (47 KU/mg).</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106621"},"PeriodicalIF":1.4,"publicationDate":"2024-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142626847","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}
Pub Date : 2024-11-05DOI: 10.1016/j.pep.2024.106620
Saira Ahmad , Qurratulann Afza Gardner , Nisar Ahmad Shakir , Sabahat Gulzar , Naseema Azim , Muhammad Akhtar
Serum amyloid A1 (SAA1) is an apolipoprotein which is involved in amyloid A amyloidosis (AA) by forming fibrils. The process of fibrillation is still being explored and holds challenges in recombinant expression and purification of SAA1. This study deals with the preferable approach for the expression and purification of SAA1 which is normally toxic and unstable to express without using any fusion-tag. Complete soluble expression of SAA1 was obtained without the use of additional tag, in terrific broth, supplemented with 3 % ethanol at 30 °C. Soluble fraction of SAA1 was initially treated with salting-out using ammonium sulphate giving 1.5 M salt concentration to avoid SAA1 protein precipitation along with unwanted proteins. The soluble fraction of SAA1 after salting-out was purified by two individual chromatographic approaches: One anion exchange and second reverse phase chromatography. The yield of purified SAA1 was 3 times greater by anion exchange than reverse phase chromatography. MALDI-TOF analysis of purified SAA1 showed 11813 Da for intact protein and proteome analysis revealed greater than 90 % sequence coverage by MASCOT. The subunit interaction showed hexamer form at basic pH which was analyzed by size exclusion chromatography. The fibrillation activity of SAA1 was found to be 10–15 times higher in basic media at 43 °C than 37 °C. Our research demonstrates successful expression and purification of wild-type human recombinant SAA1. The cost-effective radical approach employed for purification of SAA1 is crucial for thorough protein characterization particularly, mechanisms of protein aggregation involved in amyloidosis.
{"title":"Nature of recombinant human serum amyloid A1 in Escherichia coli and its preferable approach for purification","authors":"Saira Ahmad , Qurratulann Afza Gardner , Nisar Ahmad Shakir , Sabahat Gulzar , Naseema Azim , Muhammad Akhtar","doi":"10.1016/j.pep.2024.106620","DOIUrl":"10.1016/j.pep.2024.106620","url":null,"abstract":"<div><div>Serum amyloid A1 (SAA1) is an apolipoprotein which is involved in amyloid A amyloidosis (AA) by forming fibrils. The process of fibrillation is still being explored and holds challenges in recombinant expression and purification of SAA1. This study deals with the preferable approach for the expression and purification of SAA1 which is normally toxic and unstable to express without using any fusion-tag. Complete soluble expression of SAA1 was obtained without the use of additional tag, in terrific broth, supplemented with 3 % ethanol at 30 °C. Soluble fraction of SAA1 was initially treated with salting-out using ammonium sulphate giving 1.5 M salt concentration to avoid SAA1 protein precipitation along with unwanted proteins. The soluble fraction of SAA1 after salting-out was purified by two individual chromatographic approaches: One anion exchange and second reverse phase chromatography. The yield of purified SAA1 was 3 times greater by anion exchange than reverse phase chromatography. MALDI-TOF analysis of purified SAA1 showed 11813 Da for intact protein and proteome analysis revealed greater than 90 % sequence coverage by MASCOT. The subunit interaction showed hexamer form at basic pH which was analyzed by size exclusion chromatography. The fibrillation activity of SAA1 was found to be 10–15 times higher in basic media at 43 °C than 37 °C. Our research demonstrates successful expression and purification of wild-type human recombinant SAA1. The cost-effective radical approach employed for purification of SAA1 is crucial for thorough protein characterization particularly, mechanisms of protein aggregation involved in amyloidosis.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"227 ","pages":"Article 106620"},"PeriodicalIF":1.4,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142591340","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}
Pub Date : 2024-11-05DOI: 10.1016/j.pep.2024.106619
Xuebiao Zhang , Le Chen , Zhong Ni , Chao Xu , Qinyan Wu , Yiqing Zhuang
This study presents an exhaustive characterization of the enzymatic attributes and structural properties of trehalose-6-phosphate phosphatase (TPP) derived from Fusarium graminearum. Enzyme activity was evaluated through a meticulously designed enzymatic assay. The findings indicate that the molecular weight of the enzyme is approximately 99.8 kDa, with an optimal reaction temperature and pH of 40 °C and 6.5, respectively. Magnesium ions (Mg2+) markedly enhance the enzymatic activity, resulting in a specific activity of 1.795 U/μg. Kinetic analysis revealed a Km value of 0.96 μmol/L and a Vmax of 15.79 μmol/L/min. Subsequent computational analysis elucidated the three-dimensional architecture of the enzyme and identified the binding site for the substrate trehalose-6-phosphate (T6P). T6P was found to form hydrogen bonds with TPP at residues Lys754, Arg720, His665, Glu758, and Asn756. Additionally, hydrophobic interactions were observed between T6P and residues Phe802, Ile610, Asp801, Pro752, and Gly753. The binding energy calculated for the T6P-TPP complex stood at −5.7 kcal/mol.
{"title":"Trehalose-6-phosphate phosphatase expression and enzymatic properties of Fusarium graminearum","authors":"Xuebiao Zhang , Le Chen , Zhong Ni , Chao Xu , Qinyan Wu , Yiqing Zhuang","doi":"10.1016/j.pep.2024.106619","DOIUrl":"10.1016/j.pep.2024.106619","url":null,"abstract":"<div><div>This study presents an exhaustive characterization of the enzymatic attributes and structural properties of trehalose-6-phosphate phosphatase (TPP) derived from <em>Fusarium graminearum</em>. Enzyme activity was evaluated through a meticulously designed enzymatic assay. The findings indicate that the molecular weight of the enzyme is approximately 99.8 kDa, with an optimal reaction temperature and pH of 40 °C and 6.5, respectively. Magnesium ions (Mg<sup>2+</sup>) markedly enhance the enzymatic activity, resulting in a specific activity of 1.795 U/μg. Kinetic analysis revealed a <em>K</em><sub>m</sub> value of 0.96 μmol/L and a <em>V</em><sub>max</sub> of 15.79 μmol/L/min. Subsequent computational analysis elucidated the three-dimensional architecture of the enzyme and identified the binding site for the substrate trehalose-6-phosphate (T6P). T6P was found to form hydrogen bonds with TPP at residues Lys754, Arg720, His665, Glu758, and Asn756. Additionally, hydrophobic interactions were observed between T6P and residues Phe802, Ile610, Asp801, Pro752, and Gly753. The binding energy calculated for the T6P-TPP complex stood at −5.7 kcal/mol.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106619"},"PeriodicalIF":1.4,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142606002","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}
Recombinant production of lysyl endopeptidase (Lys-C) which is frequently used in proteomics is still challenging due to its complex structure. Herein, periplasmic expression and determining effective factors for recovery of the active enzyme were investigated. The codon-optimized Lys-C gene was cloned into pET26b (+) for periplasmic expression in E. coli Rosetta (DE3). The following parameters affecting expression level and activity of Lys-C were investigated including IPTG concentration (0.05–1 mM), cell density (OD600: 0.45–0.8) at induction time, presence of reducing agents (glutathione or cysteine, 0–10 mM) in culture medium or periplasmic extraction buffers, and harvesting time (6 or 20 h). Lys-C was then purified by DEAE and Ni-NTA chromatography methods. The highest expression level was obtained at 0.05 mM IPTG (5.49 %), also 8 mM cysteine, induction at OD600: 0.45 and 6 h incubation increased enzyme activity to 23.5 %, 13.3 %, and 76.4 %, respectively. The enzyme activity of Lys-C in the presence of 4 mM glutathione and extraction buffers containing 2 mM 2-mercaptoethanol (2 ME) was 81.6 % higher than the condition without reducing agents. Also, 8 mM cysteine in the culture medium and 2 mM 2 ME in extraction increased the activity up to 29.7 %. Moreover, optimization of purification process enhanced the enzyme activity from 0.217 mU to 1.76 mU. Statistical analysis showed the examined parameters significantly affected enzyme activity (p < 0.05). The presence of the reducing agents in the culture medium and extraction buffers presumably improves the Lys-C folding and increases the enzyme activity.
蛋白质组学中常用的赖氨酰内肽酶（Lys-C）结构复杂，因此其重组生产仍具有挑战性。本文研究了外质粒表达和确定回收活性酶的有效因素。将经过密码子优化的 Lys-C 基因克隆到 pET26b (+) 中，在大肠杆菌 Rosetta (DE3) 中进行质粒表达。研究了影响 Lys-C 表达水平和活性的参数，包括 IPTG 浓度（0.05-1 mM）、诱导时的细胞密度（OD600：0.45-0.8）、培养基或质外提取缓冲液中还原剂（谷胱甘肽或半胱氨酸，0-10 mM）的存在以及收获时间（6 或 20 小时）。然后用 DEAE 和 Ni-NTA 色谱法纯化 Lys-C。在 0.05 mM IPTG（5.49%）和 8 mM 半胱氨酸条件下，表达水平最高；在 OD600：0.45 和 6 小时孵育条件下，酶活性分别提高到 23.5%、13.3% 和 76.4%。在 4 mM 谷胱甘肽和含有 2 mM 2-巯基乙醇（2ME）的提取缓冲液存在下，Lys-C 的酶活性比不使用还原剂的条件下高 81.6%。此外，在培养基中加入 8 mM 半胱氨酸和在提取过程中加入 2 mM 2ME 可使活性提高 29.7%。此外，优化纯化过程可将酶活性从 0.217 mU 提高到 1.76 mU。统计分析显示，所研究的参数对酶活性有显著影响（p
{"title":"Efficient periplasmic expression of active lysyl endopeptidase and optimizing the purification methods","authors":"Zahra Pourani , Malihe Keramati , Samira Komijani , Majid Golkar , Reza Ahangari Cohan , Nastaran Mohseni , Vahideh Valizadeh","doi":"10.1016/j.pep.2024.106618","DOIUrl":"10.1016/j.pep.2024.106618","url":null,"abstract":"<div><div>Recombinant production of lysyl endopeptidase (Lys-C) which is frequently used in proteomics is still challenging due to its complex structure. Herein, periplasmic expression and determining effective factors for recovery of the active enzyme were investigated. The codon-optimized Lys-C gene was cloned into pET26b (+) for periplasmic expression in <em>E. coli</em> Rosetta (DE3). The following parameters affecting expression level and activity of Lys-C were investigated including IPTG concentration (0.05–1 mM), cell density (OD<sub>600</sub>: 0.45–0.8) at induction time, presence of reducing agents (glutathione or cysteine, 0–10 mM) in culture medium or periplasmic extraction buffers, and harvesting time (6 or 20 h). Lys-C was then purified by DEAE and Ni-NTA chromatography methods. The highest expression level was obtained at 0.05 mM IPTG (5.49 %), also 8 mM cysteine, induction at OD<sub>600:</sub> 0.45 and 6 h incubation increased enzyme activity to 23.5 %, 13.3 %, and 76.4 %, respectively. The enzyme activity of Lys-C in the presence of 4 mM glutathione and extraction buffers containing 2 mM 2-mercaptoethanol (2 ME) was 81.6 % higher than the condition without reducing agents. Also, 8 mM cysteine in the culture medium and 2 mM 2 ME in extraction increased the activity up to 29.7 %. Moreover, optimization of purification process enhanced the enzyme activity from 0.217 mU to 1.76 mU. Statistical analysis showed the examined parameters significantly affected enzyme activity (p < 0.05). The presence of the reducing agents in the culture medium and extraction buffers presumably improves the Lys-C folding and increases the enzyme activity.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106618"},"PeriodicalIF":1.4,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142591338","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}
There has been a surge in the interest to utilize plants as hosts for producing vaccine antigens. In this study, we demonstrated the successful expression of the human parvovirus B19 (B19V) capsid protein (VP2) in Nicotiana benthamiana cells. The B19V VP1 and VP2 genes were cloned under the control of estrogen-inducible promoters and transiently expressed in N. benthamiana leaves using the agroinfiltration method. The addition of estrogen significantly boosted the expression of VP2. Furthermore, codon optimization of the VP2 sequence resulted in over a 30-fold increase in its expression compared with that of the wild-type. Analysis of negatively stained samples by sucrose density gradient ultracentrifugation and electron microscopy revealed that the expressed VP2 proteins formed spherical particles with diameters of approximately 20 nm. Immunostaining analysis of protoplasts derived from VP2-expressing N. benthamiana leaves indicated that VP2 signals were predominantly localized in the cytoplasm. These findings strongly suggested that B19V VP2 assembles and formed virus-like particles (VLPs) within the cytoplasm of N. benthamiana cells, presenting a promising method for producing B19V VLPs in plant systems.
{"title":"Human parvovirus B19 virus-like particle formation in Nicotiana benthamiana","authors":"Sakika Kimura , Jiahui Ong , Atsushi Kasai , Shinji Akada , Hirotaka Ebina , Michiko Sasabe , Eiji Morita","doi":"10.1016/j.pep.2024.106616","DOIUrl":"10.1016/j.pep.2024.106616","url":null,"abstract":"<div><div>There has been a surge in the interest to utilize plants as hosts for producing vaccine antigens. In this study, we demonstrated the successful expression of the human parvovirus B19 (B19V) capsid protein (VP2) in <em>Nicotiana benthamiana</em> cells. The B19V VP1 and VP2 genes were cloned under the control of estrogen-inducible promoters and transiently expressed in <em>N. benthamiana</em> leaves using the agroinfiltration method. The addition of estrogen significantly boosted the expression of VP2. Furthermore, codon optimization of the VP2 sequence resulted in over a 30-fold increase in its expression compared with that of the wild-type. Analysis of negatively stained samples by sucrose density gradient ultracentrifugation and electron microscopy revealed that the expressed VP2 proteins formed spherical particles with diameters of approximately 20 nm. Immunostaining analysis of protoplasts derived from VP2-expressing <em>N. benthamiana</em> leaves indicated that VP2 signals were predominantly localized in the cytoplasm. These findings strongly suggested that B19V VP2 assembles and formed virus-like particles (VLPs) within the cytoplasm of <em>N. benthamiana</em> cells, presenting a promising method for producing B19V VLPs in plant systems.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106616"},"PeriodicalIF":1.4,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564768","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}
Pub Date : 2024-10-30DOI: 10.1016/j.pep.2024.106617
Jinjing Li, Baokang Wu, Yiting Ji, Shuncheng Zhang, Yuanyuan Ge, Jun Fan
Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.
{"title":"Detection and optimization of microbial expression systems for extracellular production and purification of Ca2+-responsive phase-changing annexin fusions","authors":"Jinjing Li, Baokang Wu, Yiting Ji, Shuncheng Zhang, Yuanyuan Ge, Jun Fan","doi":"10.1016/j.pep.2024.106617","DOIUrl":"10.1016/j.pep.2024.106617","url":null,"abstract":"<div><div>Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in <em>Escherichia coli</em>, <em>Bacillus subtilis</em>, and <em>Pichia pastoris</em>, and identified that the leaderless fusion protein was transported extracellularly in <em>E. coli</em> with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in <em>E. coli</em> extracellular expression systems were optimized. With coexpression of phospholipase C from <em>Bacillus cereus</em> and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from <em>E. coli</em> were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca<sup>2+</sup> and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106617"},"PeriodicalIF":1.4,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564752","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}
Nav1.7 is a eukaryotic voltage-dependent Na channel (Nav) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Nav subtype are targeted in the development of Nav1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Nav1.7 inhibitors, the chimera protein (NavAb/1.7VSD) of Nav from prokaryotic Arcobacter butzleri (NavAb), in which extracellular loops of VSD were replaced with homologous loops from Nav1.7, serves as an effective model. This is because NavAb harbors only one VSD and the biological activity of NavAb/1.7VSD was previously confirmed. To date, NavAb/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of NavAb/1.7VSD-II in Escherichia coli (E. coli). The expression of this protein in the inner membrane of E. coli was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Nav1.7-specific peptide inhibitor. The results indicate that NavAb/1.7VSD-II was functionally expressed in E. coli, providing empirical support for the discovery of new VSD-specific Nav1.7 inhibitors using the PERISS screening method.
{"title":"Functional expression of the chimera proteins of Nav1.7 and NavAb in Escherichia coli","authors":"Tomohiro Yamaguchi, Toshiaki Okada, Tadashi Kimura","doi":"10.1016/j.pep.2024.106615","DOIUrl":"10.1016/j.pep.2024.106615","url":null,"abstract":"<div><div>Na<sub>v</sub>1.7 is a eukaryotic voltage-dependent Na channel (Na<sub>v</sub>) family membrane protein and has four channel domains and four voltage sensor domains (VSD-I–IV). It is involved in pain perception, and VSDs that differ significantly by Na<sub>v</sub> subtype are targeted in the development of Na<sub>v</sub>1.7-specific inhibitors. This is expected to result in neuropathic pain treatments with fewer side effects. We previously reported on intra-periplasm secretion and selection (PERISS), a peptide drug discovery system that targets membrane proteins by co-expressing a peptide library and a target membrane protein. For PERISS screening of VSD-specific new Na<sub>v</sub>1.7 inhibitors, the chimera protein (Na<sub>v</sub>Ab/1.7VSD) of Na<sub>v</sub> from prokaryotic <em>Arcobacter butzleri</em> (Na<sub>v</sub>Ab), in which extracellular loops of VSD were replaced with homologous loops from Na<sub>v</sub>1.7, serves as an effective model. This is because Na<sub>v</sub>Ab harbors only one VSD and the biological activity of Na<sub>v</sub>Ab/1.7VSD was previously confirmed. To date, Na<sub>v</sub>Ab/1.7VSD has only been found to be expressed in insect cells. In this study, we report on the expression and channel activity of Na<sub>v</sub>Ab/1.7VSD-II in <em>Escherichia coli</em> (<em>E. coli</em>). The expression of this protein in the inner membrane of <em>E. coli</em> was confirmed by western blotting. Channel activity was assessed by measuring the channel currents of the purified recombinant proteins and inhibition using a Na<sub>v</sub>1.7-specific peptide inhibitor. The results indicate that Na<sub>v</sub>Ab/1.7VSD-II was functionally expressed in <em>E. coli</em>, providing empirical support for the discovery of new VSD-specific Na<sub>v</sub>1.7 inhibitors using the PERISS screening method.</div></div>","PeriodicalId":20757,"journal":{"name":"Protein expression and purification","volume":"226 ","pages":"Article 106615"},"PeriodicalIF":1.4,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564756","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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