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Purification his tagged protein

Weitong Pan, Yan Wang, Nan Wang
His-tagging is commonly used in fusion protein production, but the His-tag is usually prohibited in medicinal proteins and must be removed. A fragment (NCTR25 -tag) truncated from the N-terminus of human copper transporter 1 was tested for feasibility as a replacement for the His-tag in fusion proteins. The NCTR25 -tag and His-tag were separately fused to the transthyretin (TTR) protein, and the expression, affinity purification, refolding and stability of the two kinds of fusions were compared. NCTR25 fusion produced a 63% higher yield of the recombinant protein, which was purified by metal affinity chromatography with an efficiency similar to that of His-tagged protein...
August 13, 2019: Protein Expression and Purification
Hongmin Cai, Hebang Yao, Tingting Li, Yannan Tang, Dianfan Li
Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expression of the His-tagged isomerase was exceptionally high in P...
August 2, 2019: Protein Expression and Purification
Justyna Jureczek, Ralf Bergmann, Nicole Berndt, Stefanie Koristka, Alexandra Kegler, Edinson Puentes-Cala, Javier Andrés Soto, Claudia Arndt, Michael Bachmann, Anja Feldmann
Recently, we established the controllable modular UniCAR platform technology to advance the efficacy and safety of CAR T cell therapy. The UniCAR system is composed of (i) target modules (TMs) and (ii) UniCAR armed T cells. TMs are bispecific molecules that are able to bind to the tumor cell surface and simultaneously to UniCAR T cells. For interaction with UniCAR T cells, TMs contain a peptide epitope sequence which is recognised by UniCAR T cells. So far, a series of TMs against a variety of tumor targets including against the prostate stem cell antigen (PSCA) were constructed and functionally characterised...
July 22, 2019: Scientific Reports
Yaprak Aslantas, Nur Basak Surmeli
Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of "unactivated" carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius , which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH...
2019: Bioinorganic Chemistry and Applications
Yi-Ling Bai, Md Shahed-Al-Mahmud, Karuppuchamy Selvaprakash, Nien-Tsung Lin, Yu-Chie Chen
Acinetobacter baumannii strains are common nosocomial pathogens that can cause infections and can easily become resistant to antibiotics. Thus, analytical methods that can be used to rapidly identify A. baumannii from complex samples should be developed. Tail fiber proteins derived from the tail fibers of bacteriophages can recognize specific bacterial surface polysaccharides. For example, recombinant tail proteins, such as TF2 and TF6 derived from the tail fibers of bacteriophages ϕAB2 and ϕAB6, can recognize A...
July 10, 2019: Analytical Chemistry
Hui Xu, Thomas Clairfeuille, Christine C Jao, Hoangdung Ho, Zachary Sweeney, Jian Payandeh, Christopher M Koth
Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets...
2019: Methods in Molecular Biology
Yang Zhou, Xiaofeng Li, Dandan Yan, Frank Addai Peprah, Xingqi Ji, Emmanuella Esi Fletcher, Yanwei Wang, Yingying Wang, Jie Gu, Feng Lin, Haifeng Shi
Background: In the enzymatic conversion of biomass, it becomes an important issue to efficiently and cost-effectively degrade cellulose into fermentable glucose. β-Glucosidase (Bgluc), an essential member of cellulases, plays a critical role in cellulosic biomass degradation. The difficulty in improving the stability of Bgluc has been a bottleneck in the enzyme-dependent cellulose degradation. The traditional method of protein purification, however, leads to higher production cost and a decrease in activity...
2019: Biotechnology for Biofuels
Lei Zhang, Linchao Lei, Guangya Zhang, Xialan Li
A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one...
May 25, 2019: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Dan-Yang Shi, Fu-Jia Liu, Yun-Yun Mao, Rong-Tian Cui, Jian-Sheng Lu, Yun-Zhou Yu, Xiao-Jie Dong, Zhi-Xin Yang, Zhi-Wei Sun, Xiao-Bin Pang
Botulinum neurotoxins (BoNTs) are among the most toxic proteins. Vaccination is an effective strategy to prevent botulism. To generate a vaccine suitable for human use, a recombinant non-His-tagged isoform of the Hc domain of botulinum neurotoxin serotype E (rEHc) was expressed in Escherichia coli and purified by sequential chromatography. The immunogenicity of rEHc was evaluated in mice and dose- and time-dependent immune responses were observed in both antibody titers and protective potency. Then, the pilot-scale expression and purification of rEHc were performed and its immunological activity was characterized...
June 18, 2019: Human Vaccines & Immunotherapeutics
Laura Del Amo-Maestro, Laura Marino-Puertas, Theodoros Goulas, F Xavier Gomis-Rüth
Transforming growth factor β is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFβ1-TGFβ3) engaged in signaling functions through binding of cognate TGFβ receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFβs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFβs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins...
June 17, 2019: Scientific Reports
Yuanzhong Yang, Khosse Mitri, Chunfang Zhang, Reinhard I Boysen, Milton T W Hearn
Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+ -1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6 -tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates...
June 3, 2019: Protein Expression and Purification
María Victoria Humbert
Modern DNA recombinant techniques and major advances in genetic engineering have resulted in the development of bacterial expression systems that guarantee an unlimited supply of valuable proteins that have potential clinical or industrial use, but which are often limited by their low natural availability. This chapter provides the reader with a general scheme to clone, express, and purify native histidine (His)-tagged proteins in the desired quantity and quality required for its intended use, and reviews the most important factors affecting the production of recombinant proteins in a soluble form...
2019: Methods in Molecular Biology
M Ebrahimi, H Hamidinejat, M R Tanabandeh, M H Razi Jalali, A Rasouli
Diversity among the pathogenic strains of Theileria equi (T. equi), a major agent of equine piroplasmosis, can affect the appropriate detection of parasite and host immunization. Production of recombinant surface proteins from an infected horse in natural endemic area provides a reliable tool for immunodiagnosis of parasite. Regarding this, the present study was targeted toward the cloning, expression, and purification of the immunogenic regions of equine merozoite antigen 1 (EMA-1 gene), as one of the most important immunodominant surface proteins in T...
December 2018: Archives of Razi Institute
Shang Jia, Dan He, Christopher J Chang
Site-selective bioconjugation to native protein residues is a powerful tool for protein functionalization, with cysteine and lysine side chains being the most common points for attachment owing to their high nucleophilicity. We now report a strategy for histidine modification using thiophosphorodichloridate reagents that mimic post-translational histidine phosphorylation, enabling fast and selective labeling of protein histidines under mild conditions where various payloads can be introduced via copper-assisted alkyne-azide cycloaddition (CuAAC) chemistry...
April 24, 2019: Journal of the American Chemical Society
Rajani Adiga, Mustafa Al-Adhami, Abhay Andar, Shayan Borhani, Sheniqua Brown, David Burgenson, Merideth A Cooper, Sevda Deldari, Douglas D Frey, Xudong Ge, Hui Guo, Chandrasekhar Gurramkonda, Penny Jensen, Yordan Kostov, William LaCourse, Yang Liu, Antonio Moreira, KarunaSri Mupparapu, Chariz Peñalber-Johnstone, Manohar Pilli, Benjamin Punshon-Smith, Aniruddha Rao, Govind Rao, Priyanka Rauniyar, Sergei Snovida, Kanika Taurani, Dagmawi Tilahun, Leah Tolosa, Michael Tolosa, Kevin Tran, Krishna Vattem, Sudha Veeraraghavan, Brandon Wagner, Joshua Wilhide, David W Wood, Adil Zuber
Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins...
September 2018: Nature biomedical engineering
Shing-Ling Tsai, Yung-Chieh Chang, Sailu Sarvagalla, Shuying Wang, Mohane Selvaraj Coumar, Chun Hei Antonio Cheung
Survivin is a well-known inhibitor-of-apoptosis proteins family member and a promising molecular target for anti-cancer treatment. However, it is widely accepted that survivin is only a "semi-druggable" target and development of survivin-specific small molecule inhibitors has shown to be difficult. In this study, we demonstrated that a histidine-tagged survivin T34A-C84A mutated protein (T34A-C84A-dNSur-His) can be produced using a bacterial recombinant protein expression system [E. coli ArcticExpress (DE3) cells] and solubilized using 1% (w/v) Sarkosyl...
April 17, 2019: Protein Expression and Purification
Jian Gao, Ding Zhao
Multi-epitope recombinant diagnostic antigen (designated 'B102') of Mycobacterium tuberculosis (Mtb) was prepared and evaluated as a serological diagnostic antigen. With TRX at the N-terminal and His tag at the C-terminal, the multi-epitope Mtb recombinant diagnostic antigen including 11 predicted B-cell epitopes from 6 Mtb antigens (PstS1, ESAT6, CFP10, Ag85B, Ag85A and PPE54) was expressed in Escherichia coli BL21 (DE3) and purified by Ni²⁺-Chelating affinity and DEAE anion exchange chromatography. Based on the antigenicity of B102 confirmed in Western blotting analysis, we constructed and evaluated a double-antigen sandwich ELISA for diagnosis of Mtb infection...
April 25, 2019: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
X Li, M Liu, X Bai, Y Li, Y Zhao, S Wang, J Wang
Osteocalcin is a major non-collagenous component of the bone extracellular matrix and is considered to be an indicative factor of osteoblast differentiation. In the present study, we detected osteocalcin expression in different antler areas and growth phases by immunohisto- chemistry. Osteocalcin was highly expressed in all areas during the mineralization period and in mesenchymal cell and chondrocyte areas during the rapid growth period. The nucleotide sequence of the osteocalcin gene in sika deer antler was determined...
March 2019: Polish Journal of Veterinary Sciences
Irsyad N A Khairil Anuar, Anusuya Banerjee, Anthony H Keeble, Alberto Carella, Georgi I Nikov, Mark Howarth
Peptide tags are a key resource, introducing minimal change while enabling a consistent process to purify diverse proteins. However, peptide tags often provide minimal benefit post-purification. We previously designed SpyTag, forming an irreversible bond with its protein partner SpyCatcher. SpyTag provides an easy route to anchor, bridge or multimerize proteins. Here we establish Spy&Go, enabling protein purification using SpyTag. Through rational engineering we generated SpyDock, which captures SpyTag-fusions and allows efficient elution...
April 15, 2019: Nature Communications
Yurie Kinoshita, Jian Xu, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Jae Man Lee
Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph...
March 24, 2019: Protein Expression and Purification
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