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Protein Expression and Purification

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https://www.readbyqxmd.com/read/28526454/generation-and-characterization-of-caprine-chymosin-in-corn-seed
#1
Wen-Guo Liu, Yun-Peng Wang, Zhi-Jun Zhang, Min Wang, Qing-Xue Lv, Hong-Wei Liu, Ling-Cong Meng, Ming Lu
Chymosin is widely used in the dairy industry, and much is produced through recombinant DNA in organisms such as bacteria and tobacco. In this study, we used a new transgenic method to express caprine chymosin in corn seeds with lower cost and better storage capability. The recombinant chymosin protein was successfully expressed at an average level of 0.37 mg/g dry weight, which is 0.27% of the total soluble protein in the corn seed. Prochymosin can be activated to produce a chymosin protein with the ability to induce clotting in milk, similar to the commercial protein...
May 16, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28506644/affinity-purification-of-car9-tagged-proteins-on-silica-matrices-optimization-of-a-rapid-and-inexpensive-protein-purification-technology
#2
Jessica Soto-Rodríguez, Brandon L Coyle, Ariana Samuelson, Kannan Aravagiri, François Baneyx
Car9, a dodecapeptide identified by cell surface display for its ability to bind to the edge of carbonaceous materials, also binds to silica with high affinity. The interaction can be disrupted with l-lysine or l-arginine, enabling a broad range of technological applications. Previously, we reported that C-terminal Car9 extensions support efficient protein purification on underivatized silica. Here, we show that the Car9 tag is functional and TEV protease-excisable when fused to the N-termini of target proteins, and that it supports affinity purification under denaturing conditions, albeit with reduced yields...
May 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28502666/purification-and-characterisation-of-the-fission-yeast-ndc80-complex
#3
Yuzy Matsuo, Sebastian P Maurer, Thomas Surrey, Takashi Toda
The Ndc80 complex is a conserved outer kinetochore protein complex consisting of Ndc80 (Hec1), Nuf2, Spc24 and Spc25. This complex comprises a major, if not the sole, platform with which the plus ends of the spindle microtubules directly interact. In fission yeast, several studies indicate that multiple microtubule-associated proteins including the Dis1/chTOG microtubule polymerase and the Mal3/EB1 microtubule plus-end tracking protein directly or indirectly bind Ndc80, thereby ensuring stable kinetochore-microtubule attachment...
May 11, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28499579/expression-of-recombinant-truncated-domains-of-mucus-binding-mub-protein-of-lactobacillus-plantarum-in-soluble-and-biologically-active-form
#4
Kumar Siddharth Singh, Ritu Choudhary, Sonu Bisht, Sunita Grover, Sudarshan Kumar, Ashok Kumar Mohanty, Jai Kumar Kaushik
No abstract text is available yet for this article.
May 10, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28487257/functional-efficacy-of-human-recombinant-fgf-2s-tagged-with-his-6-and-his-asn-6-at-the-n-and-c-termini-in-human-gingival-fibroblast-and-periodontal-ligament-derived-cells
#5
Ji-Hye Lee, Ji-Eun Lee, Kyung-Jung Kang, Young-Joo Jang
Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H6) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN6) is also efficient for purification as it is easily exposed on the surface of the protein...
May 6, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28476488/improving-pertuzumab-production-by-gene-optimization-and-proper-signal-peptide-selection
#6
Amin Ramezani, Elham Mahmoudi Maymand, Mahsa Yazdanpanah-Samani, Ahmad Hosseini, Fatemeh Sadat Toghraie, Abbas Ghaderi
Using proper signal peptide and codon optimization are important factors that must be considered when designing the vector to increase protein expression in Chinese Hamster Ovary (CHO) cells. The aim of the present study is to investigate how to enhance Pertuzumab production through heavy and light chain coding gene optimization and proper signal peptide selection. First, CHO-K1 cells were transiently transfected with whole-antibody-gene-optimized, variable-regions-optimized and non-optimized constructs and then we employed five different signal peptides to improve the secretion efficiency of Pertuzumab...
May 3, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28465181/expression-purification-and-characterization-of-recombinant-8%C3%A2-kda-gelsolin-fragment
#7
Qing Zhang, Weijie Lu, Lina Ji, Zi-Chun Hua
A mutation (D187N/Y) in human plasma gelsolin (GSN) leads to the generation of an 8 kDa GSN fragment (8 kDa-GSN), and consequently causes the familial amyloidosis of Finnish type. Because of its faster kinetics of amyloid formation under physiologically relevant conditions, 8 kDa-GSN is used to explore gelsolin amyloidosis and screen small molecules that can disaggregate amyloids. However, the synthetic 8 kDa-GSN is expensive, and substantial quantities of 8 kDa-GSN are needed for the screen. Here we report a study to obtain recombinant 8 kDa-GSN with high yield from Escherichia coli...
April 29, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28458053/overexpression-of-ebola-virus-envelope-gp1-protein
#8
Zhongcheng Zou, John Misasi, Nancy Sullivan, Peter D Sun
Ebola virus uses its envelope GP1 and GP2 for viral attachment and entry into host cells. Due to technical difficulty expressing full-length envelope, many structural and functional studies of Ebola envelope protein have been carried out primarily using GP1 lacking its mucin-like domain. As a result, the viral invasion mechanisms involving the mucin-like domain are not fully understood. To elucidate the role of the mucin-like domain of GP1 in Ebola-host attachment and infection and to facilitate vaccine development, we constructed a GP1 expression vector containing the entire attachment region (1-496)...
April 27, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28458052/production-of-monoclonal-antibodies-and-development-of-a-quantitative-immuno-polymerase-chain-reaction-assay-to-detect-and-quantify-recombinant-glutathione-s-transferase
#9
Abud Je, Luque Eh, Ramos Jg, Rodriguez Ha
GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST)...
April 27, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28442431/reducing-isoform-complexity-of-human-tetraspanins-by-optimized-expression-in-dictyostelium-discoideum-enables-high-throughput-functional-read-out
#10
Tineke Scheltz, Julia von Bülow, Eric Beitz
The human tetraspanin family of scaffold proteins comprises 33 isoforms. Being integral membrane proteins, they organize a so-called tetraspanin web via homomeric and heteromeric protein-protein interactions with integrins, immunoglobulins, growth factors, receptor tyrosine kinases, proteases, signaling proteins, and viral capsid proteins. Tetraspanins promote cellular effects, such as adhesion, migration, invasion, signaling, membrane fusion, protein trafficking, cancer progression, and infections. The ubiquitous expression of multiple tetraspanin isoforms and partner proteins hampers specific interaction studies...
April 22, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28442430/design-and-purification-of-active-truncated-phosphoinositide-3-kinase-gamma-protein-constructs-for-structural-studies
#11
A Vujičić Žagar, L Scapozza, O Vadas
Phosphoinositide 3-kinase gamma (PI3Kγ) is a lipid kinase that plays a crucial role in cell migration, chemotaxis, oxidative burst and myocardial contractility. It is activated downstream of G protein-coupled receptors (GPCRs) and small GTPases of Ras superfamily. PI3Kγ is a heterodimer composed of a catalytic and a regulatory subunit that is expressed mostly in hematopoietic cells and in the heart. Although it has attracted a lot of attention because of its link with tumor inflammation and heart diseases, its regulation is still not fully understood...
April 22, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28438686/expression-purification-and-molecular-characterization-of-a-novel-endoglucanase-protein-from-bacillus-subtilis-sb13
#12
Xuefang Guan, Penglian Chen, Qingxian Xu, Lei Qian, Juqing Huang, Bin Lin
Bacillus subtilis strain SB13 which is isolated in our previous work was confirmed to produce endoglucanase. In this study, a novel endoglucanase gene (accession number: KX576676) was identified and cloned from SB13. Compared with other consensus sequence of reported endoglucanase genes in the GenBank database, this gene displays five differences (including T740C,A874G,A983G, T1210G and T1301C), which leading to five amino acid changes. Homology modeling has indicated that these five changes were located in the α-helix and random coil regions of the glycosyl hydrolase family 5 (GH5) domain, the random coil and β-sandwich of the type 3 carbohydrate-binding module (CBM3) domain, and the random coil domain...
April 21, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28428153/development-of-polyol-responsive-antibody-mimetics-for-single-step-protein-purification
#13
Richard J Suderman, Daren A Rice, Shane D Gibson, Eric J Strick, David M Chao
The purification of functional proteins is a critical pre-requisite for many experimental assays. Immunoaffinity chromatography, one of the fastest and most efficient purification procedures available, is often limited by elution conditions that disrupt structure and destroy enzymatic activity. To address this limitation, we developed polyol-responsive antibody mimetics, termed nanoCLAMPs, based on a 16 kDa carbohydrate binding module domain from Clostridium perfringens hyaluronidase. nanoCLAMPs bind targets with nanomolar affinity and high selectivity yet release their targets when exposed to a neutral polyol-containing buffer, a composition others have shown to preserve quaternary structure and enzymatic activity...
April 17, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28419821/expression-of-the-enzymatically-active-legumain-like-cysteine-proteinase-tvlegu-1-of-trichomonas-vaginalis-in-pichia-pastoris
#14
Gerardo Reséndiz-Cardiel, Rossana Arroyo, Jaime Ortega-López
The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter...
April 15, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28414067/effective-strategies-for-host-cell-protein-clearance-in-downstream-processing-of-monoclonal-antibodies-and-fc-fusion-proteins
#15
REVIEW
Yifeng Li
Recombinant therapeutic proteins are typically produced through cell culture process. Host cell proteins (HCPs) are endogenous proteins derived from the host cells used for such bioproduction. HCPs form a major class of process-related impurities and even at low levels they can potentially compromise the safety and efficacy of biopharmaceuticals. Therefore, they need to be adequately removed via the downstream process. HCPs are complex mixtures with diverse physiochemical properties, and certain subpopulations can bind to the intended product...
April 13, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28410993/development-of-a-novel-recombinant-lhrh-fusion-protein-for-therapy-of-androgen-and-estrogen-dependent-cancers
#16
Jagdish C Gupta, Rohit S Hada, P Sahai, G P Talwar
LHRH based vaccines are promising candidates for therapy of androgen and estrogen dependent cancers. We report in this communication development of a novel recombinant protein vaccine candidate against LHRH. A synthetic gene was designed in which the codon sequence in the LHRH decapeptide was modified by substituting the codon for 6-glycine with that of l-leucine. Further the LHRH(6leu) gene was linked to heat-labile enterotoxin of E. coli (LTB) as carrier. This LHRH(6leu)-LTB gene was cloned into a prokaryotic expression vector under the control of inducible and strong bacteriophage T7 promoter to over-express LHRH(leu) fused to LTB as recombinant protein in E...
April 12, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28400295/phic31-integrase-can-improve-the-efficiency-of-different-construct-designs-for-monoclonal-antibody-expression-in-cho-cells
#17
Maryam Ahmadi, Fereidoun Mahboudi, Samira Ahmadi, Saeedeh Ebadat, Fatemeh Nematpour, Mohammad Reza Akbari Eidgahi, Fatemeh Davami
OBJECTIVES: Several types of expression vectors have been used for recombinant protein expression in Chinese hamster ovary cells (CHO) which usually result in variable and unstable levels of expression. METHODS AND RESULTS: In this study, we have compared the mAb0014 expression level of single ORF/IRES vector and dual ORF vector in the presence and absence of phiC31 integrase targeting system. Both expression vectors contain an elongation factor 1α (EF1α) promoter upstream of LC and harboring an attB site...
April 9, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28400296/recombinant-expression-and-purification-of-the-rna-binding-larp6-proteins-from-fish-genetic-model-organisms
#18
José M Castro, Daniel A Horn, Karen A Lewis
The RNA-binding proteins that comprise the La-related protein (LARP) superfamily have been implicated in a wide range of cellular functions, from tRNA maturation to regulation of protein synthesis. To more expansively characterize the biological function of the LARP6 subfamily, we have recombinantly expressed the full-length LARP6 proteins from two teleost fish, platyfish (Xiphophorus maculatus) and zebrafish (Danio rerio). The yields of the recombinant proteins were enhanced to >2 mg/L using a tandem approach of an N-terminal His6-SUMO tag and an iterative solubility screening assay to identify structurally stabilizing buffer components...
April 8, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28392342/exploitation-of-human-cd99-expressing-mouse-myeloma-cells-as-immunogen-for-production-of-mouse-specific-polyclonal-antibodies
#19
Nuchjira Takheaw, Witida Laopajon, Kantinan Chuensirikulchai, Watchara Kasinrerk, Supansa Pata
In this study, we describe the application of a molecular biology technique for the production of mouse polyclonal antibodies (pAbs) specific to human cell surface molecules. Production of the pAb specific to the human CD99 surface molecule was used as the study model. The retroviral expression system was employed to generate human CD99 expressing mouse myeloma cells. After cell sorting and single cell cloning, a myeloma clone which stably expressed high levels of human CD99 on its surface was established. The human CD99 expressing mouse myeloma cells were then used as the immunogen for immunization of BALB/c mice...
April 6, 2017: Protein Expression and Purification
https://www.readbyqxmd.com/read/28389350/process-development-for-production-and-purification-of-the-schistosoma-mansoni-sm14-antigen
#20
Leonardo Damasceno, Gerd Ritter, Carl A Batt
The trematode Schistosoma mansoni Sm14 antigen was expressed in the yeast Pichia pastoris and secreted into the culture medium at yields of approximately 250 mg L(-1). Sm14 belongs to a family of fatty-acid binding proteins and appears to play an important role in uptake, transport, and compartmentalization of lipids in S. mansoni and it is a potential vaccine candidate in both humans and domesticated animals. The Sm14 gene was codon-optimized for expression in P. pastoris, and placed under transcription of the strong methanol inducible AOX1 promoter...
April 5, 2017: Protein Expression and Purification
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