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Protein Expression and Purification

Tatsuya Kato, Moeko Hasegawa, Takeshi Yamamoto, Takatsugu Miyazaki, Ryosuke Suzuki, Takaji Wakita, Tetsuro Suzuki, Enoch Y Park
It has been shown that the single-domain intrabody 2H9-L against the hepatitis C virus (HCV) capsid (core) protein inhibits the viral propagation and NF-κB promoter activity induced by the HCV core. In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1-123 amino acid residues (C123), 1-152 amino acid residues (C152), 1-177 amino acid residues (C177) and 1-191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E...
May 17, 2018: Protein Expression and Purification
Richard R Burgess
Through years of research, teaching, troubleshooting, and editing, I have developed a solid working understanding of many aspects of protein purification and protein biochemistry. I have also found that many researchers do not understand the basics, let alone the pitfalls, of the techniques they use. In the case of size exclusion chromatography (SEC), I summarize below some of the rules of thumb, suggestions, limitations and troubleshooting I have found most useful in helping my students and colleagues carry out successful SEC...
May 16, 2018: Protein Expression and Purification
Mehrnoosh Fathi-Roudsari, Nader Maghsoudi, Amirhossein Maghsoudi, Sepideh Niazi, Morvarid Soleiman
Reteplase is a third generation tissue plasminogen activator (tPA) with a modified structure and prolonged half-life in comparison to native tPA. As a non-glycosylated protein, reteplase is expressed in Escherichia coli. Due to presence of several disulfide bonds, high level production of reteplase is complicated and needs extra steps for conversion to biologically active form. Auto-induction represents a method for high-yield growth of bacterial cells and higher expression of recombinant proteins. Here we have tried to optimize the auto-induction procedure for soluble and active expression of reteplase in E...
May 15, 2018: Protein Expression and Purification
Yanan Guo, Miao Yu, Na Jing, Shoutao Zhang
Embryonic stem cells and induced pluripotent stem cells depend on one of cytokines called leukemia inhibitory factor (LIF) to retain their undifferentiated state and pluripotency. Nevertheless, further progresses of stem cell scientific investigation and its possible application are limited owing to the expense of commercial LIF. Here we introduced a simple, practical and high level expression of MBP-mouse LIF through Escherichia coli system which was bioactive. The mLIF cDNA was inserted into vector of pMAL-C2X in order to generate N-terminal MBP-mLIF recombinant proteins in the cytoplasm of Escherichia coli...
May 11, 2018: Protein Expression and Purification
Shengjun Wang, Yaoguang Wang, Peng George Wang, Min Chen, Yun Kong
The Ala-Pro-rich Antigen (Apa) from Mycobacterium tuberculosis is a mannosylated protein with immunogenic and antigenic properties. The O-mannosylation is essential for its biological function in the process of colonization and invasion of host cells by M. tuberculosis. In this work, the gene encoding Apa was cloned from M. tuberculosis and expressed in Pichia pastoris GS115. In shake-flasks, the recombinant Apa was secreted into the culture media and purified with a yield of 0.6 g/L. Both N- and O-glycans were found in recombinant Apa...
May 9, 2018: Protein Expression and Purification
Peter Rehbein, Harald Schwalbe
Production of recombinant prion proteins is of crucial relevance in food technology (analytical standards, assay development) but also in basic research, most importantly structural biology (NMR, X-ray diffraction). Structural approaches conveniently allow for sophisticated investigation of prion disease pathogenesis, but usually require large amounts of sample material. Recently, working with recombinant prion proteins has been recategorized to biosafety levels > S1 as infectious prions may readily be generated de novo and become airborne via aerosols...
May 8, 2018: Protein Expression and Purification
Tian Zhao, Zhipeng Li, Zhouliang Guo, Ali Wang, Zhenxing Liu, Qing Zhao, Yuyin Li, Edward A McKenzie, Aipo Diao
Legumain (LGMN) is a lysosomal protease that can specifically hydrolyze proteins after carboxyl-terminal asparagine residues. It has been reported that Legumain is highly expressed in many human tumors and promotes the migratory and invasive activity of cancer cells. Due to the limitation of an abundant and affordable source of endogenous active Legumain for further function studies, we produced the recombinant protein in Pichia pastoris. The pPICZα-LGMN expression plasmid was constructed and transformed into Pichia pastoris strain and positive recombinants were identified...
May 7, 2018: Protein Expression and Purification
Natthaya Mangkorn, Pattanop Kanokratana, Niran Roongsawang, Navadol Laosiripojana, Verawat Champreda
Alcohol oxidase (AOX) functions in oxidation of primary alcohols into the corresponding aldehydes with potential on catalyzing synthesis reactions in chemical industry. In this study, AOX from a thermotolerant methylotrophic yeast, Ogataea thermomethanolica (OthAOX) was purified to high homogeneity using a single step chromatographic separation on a DEAE-Sepharose column. The purified OthAOX had a specific activity of 15.34 U/mg with 77.5% recovery yield. The enzyme worked optimally at 50 °C in an alkaline range (pH 9...
May 5, 2018: Protein Expression and Purification
Sudarat Hadpech, Wannarat Jinathep, Somphot Saoin, Weeraya Thongkum, Koollawat Chupradit, Umpa Yasamut, Sutpirat Moonmuang, Chatchai Tayapiwatana
The requirement for reliable bicistronic or multicistronic vectors in gene delivery systems is at the forefront of bio/biomedical technology. A method that provides an efficient co-expression of multiple heterologous proteins would be valuable for many applications, especially in medical science for treating various types of disease. In this study, we designed and constructed a bicistronic expression vector using a self-cleaving 2A peptide derived from a virus of the insect Thosea asigna (T2A). This exhibited the most efficient cleavage of the 2A sequence...
May 4, 2018: Protein Expression and Purification
Zhenzhan Chang, Xiaoqiang Wang, Risheng Wei, Zhouying Liu, Hong Shan, Guizhen Fan, Hongli Hu
Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus...
May 3, 2018: Protein Expression and Purification
Aline Vianna Bernardi, Paula Fagundes de Gouvêa, Luis Eduardo Gerolamo, Deborah Kimie Yonamine, Laís de Lourdes de Lima Balico, Sergio Akira Uyemura, Taisa Magnani Dinamarco
A gene encoding an endo-1,4-β-glucanase (Afu6g01800) from A. fumigatus was cloned into the vector pET-28a(+) and expressed in the E. coli strain RosettaTM (DE3) pLysS. Sequence analysis indicated that the enzyme Af-EGL7 belonged to the GH7 family. The gene Af-egl7 encoded a protein comprising 460 amino acids, with a CBM1 domain at residues 424-460 and molecular mass of 52 kDa, as estimated by SDS-PAGE. This enzyme was optimally active at pH and temperatures ranging from 4.5 to 5.5 and from 40 to 60 °C, respectively...
April 28, 2018: Protein Expression and Purification
Mathias Kahl, Florian Settele, Paul Knick, Ulrich Haupts, Eva Bosse-Doenecke
Protein based binding molecules have a broad applicability from therapeutic to technical use. Monoclonal antibodies represent the major class of this type of agents complemented by innovative approaches using scaffold proteins with tailor-made properties. Various concepts for new formats combining antibody chains or antibody fragments and fusions with other entities have been developed recently. This strategy opens up options to design molecules with biophysical, biochemical and pharmacological characteristics in a broad range while simultaneously addressing several targets or epitopes...
April 25, 2018: Protein Expression and Purification
Satoru Shimada, Shintaro Maeda, Masahide Hikita, Kaoru Mieda-Higa, Shigefumi Uene, Yukiko Nariai, Kyoko Shinzawa-Itoh
Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46...
April 24, 2018: Protein Expression and Purification
Wenqian Li, Hao Shi, Huaihai Ding, Liangliang Wang, Yu Zhang, Xun Li, Fei Wang
This paper first describes characterization of two thermostable esterases (ThLip1 and ThLip2) from the thermophilic bacterium Thermoanaerobacterium thermosaccharolyticum DSM 571. The recombinant esterase ThLip1 was active at 80 °C, pH 6.5 and maintained approx. 85% of original activity after 2 h incubation at 75 °C. Kinetic parameters, Km , Vmax and kcat /Km for 4-Nitrophenyl caprylate (pNPC) were 3.52 ± 0.47 mM, 191.18 ± 1.82 μmol min-1 mg-1 and 20.80 ± 0.07 mM-1  s-1 , respectively...
April 20, 2018: Protein Expression and Purification
Guan-Qun Chen, Di Zhang, Xiao-Hui Shen
Plant cystatins are involved in the regulation of protein turnover and play important roles in defense mechanisms. We cloned the ApCystatin gene from Agapanthus praecox ssp. orientalis, a famous ornamental and medical plant. The complete cDNA sequence of ApCystatin is comprised of 1446 nucleotides with a 420 bp ORF encoding 140 amino acids. The mRNA level of ApCystatin was significantly up-regulated under various abiotic stress, such as salt, osmosis, oxidative and cold stresses, which suggested that ApCystatin participated in the plant's resistance to stress...
April 20, 2018: Protein Expression and Purification
Shijie Li, Linlin Li, Zhuo Chen, Guangpu Xue, Longguang Jiang, Ke Zheng, Jincan Chen, Rui Li, Cai Yuan, Mingdong Huang
Plasma-derived human serum albumin (pHSA) has important applications in many clinical indications, including blood loss, serious burn, or hemorrhagic shock. The limited supply and potential infectious pathogen contamination of pHSA have stimulated the development of recombinant human serum albumin (rHSA). However, rHSA often entraps endogenous or exogenous impurities, including color pigments and polysaccharides. Therefore, the purification of rHSA to high purity remains the bottleneck in large-scale production of rHSA...
April 17, 2018: Protein Expression and Purification
Yaru Yan, Xiaoqing Liu, Qingbin Li, Xiaoyu Chu, Jian Tian, Ningfeng Wu
In the previous study, the results on two interesting egfp genes indicated that the expressed eGFP production of egfp-codon containing multiple rare codons was 2.3-fold than that of egfp-genscript with mainly high-frequency-usage codons. Therefore, the rare codons also play important roles for the functional expression of genes and it is interesting to know which rare codons in the egfp affect the functional expression of eGFP. In this study, the structure-guided SCHEMA recombination method and site-specific mutagenesis were proposed to detect the contribution of the rare codons on the functional expression of eGFP...
April 17, 2018: Protein Expression and Purification
Alexander J Winton, Janae L Baptiste, Mark A Allen
Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials...
April 16, 2018: Protein Expression and Purification
Dengyue Sun, Dengke Gao, Panpan Xu, Qianqian Guo, Zhangliang Zhu, Xiaotao Cheng, Song Bai, Hui-Min Qin, Fuping Lu
Hydroxy amino acids are produced by Fe(II)/αKG-dependent dioxygenases and used widely as medicinal intermediates for chemical synthesis. A novel l-leucine 5-hydroxylase gene from Nostoc piscinale (NpLDO) was cloned into pET28a (+), pColdI and pQE-80 L plasmids. Using a two-step purification process (Ni-affinity chromatography and gel filtration), highly purified recombinant NpLDO was obtained. Recombinant NpLDO displayed unexpectedly high sulfoxidation activity toward l-methionine. The reaction products were analyzed by high-performance liquid chromatography...
April 16, 2018: Protein Expression and Purification
Sujeewa D Wijesuriya, Elizabeth Pongo, Milan Tomic, Fangjiu Zhang, Consuelo Garcia-Rodriquez, Fraser Conrad, Shauna Farr-Jones, James D Marks, Arnold H Horwitz
Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies...
April 12, 2018: Protein Expression and Purification
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