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Protein Expression and Purification

Junfeng Zhang, Liping He, Yougen Wu, Wentin Ma, He Chen, Zhouchen Ye
A proteomic approach was used to understand the molecular mechanisms underlying obstacles to the continuous cropping of Pogostemon cablin. We examined differences in protein abundance between control (CK) and continuously cropped (TR) P. cablin leaves at different time points (90, 150, and 210 days after culture). Comparative analysis by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) revealed 183 differentially expressed protein spots, of which 87 proteins or isoforms were identified using mass spectrometry...
July 12, 2018: Protein Expression and Purification
Sarah E Hopson, Martin J Thompson
The human polybromo-1 protein (BAF180) is a known driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. BAF180 is the chromatin-targeting subunit of the PBAF complex. BAF180 has six bromodomains, two BAH domains, and one HMG box. Bromodomains are known to recognize acetylated-lysines on histones and play a role in nucleosome recognition. BAH domains are required for ubiquitination of PCNA, a key regulator of DNA damage. The putative HMG box, if functional, may be involved in DNA-binding...
July 12, 2018: Protein Expression and Purification
Ruixue Wang, Qinhuai Lai, Ying Lu, Yan Zhou, Liangze Tang, Yiran Tao, Yuqin Yao, Lin Yu, Yu Liu, Yuxi Wang, Ruirui Zhang, Xiaohua Jiang, Lantu Gou, Jinliang Yang
5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen...
July 5, 2018: Protein Expression and Purification
Michael Chow, Meropi Sklepari, Lorenzo Frigerio, Ann M Dixon
Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in length but all contain a conserved C-terminal reticulon homology domain (RHD) that associates with membranes. An understanding of the structure and membrane interactions of RHDs is key to unlocking their mechanism of function, however no three-dimensional structure has been solved...
June 30, 2018: Protein Expression and Purification
Jian Ma, Yunpeng Wang, Nuo Xu, Libo Jin, Jia Liu, Shaochen Xing, Xiaokun Li
Factor H binding protein (fHbp) is the most promising vaccine candidate against serogroup B of Neisseria meningitidis which is a major cause of morbidity and mortality in children. In order to facilitate large scale production of a commercial vaccine, we previously used transgenic Arabidopsis thaliana, but plant-derived fHbp is still far away from a commercial vaccine due to less biomass production. Herein, we presented an alternative route for the production of recombinant fHbp from the seeds of transgenic rice...
June 25, 2018: Protein Expression and Purification
Suvash Chandra Ojha, Chompounoot Imtong, Kanungsuk Meetum, Somsri Sakdee, Gerd Katzenmeier, Chanan Angsuthanasombat
Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn2+ -dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn2+ ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni2+ affinity chromatography (IMAC)...
June 23, 2018: Protein Expression and Purification
Ulrike Blaschke, Beneditta Suwono, Sachli Zafari, Ingo Ebersberger, Evelyn Skiebe, Cy M Jeffries, Dmitri I Svergun, Gottfried Wilharm
Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978...
June 22, 2018: Protein Expression and Purification
Lauren Procter, Carissa Grose, Dominic Esposito
Protein prenylation is a vital eukaryotic post-translational modification which permits interaction of proteins with cellular membranes. Prenylated proteins are involved in a number of human diseases, and play a major role in cancers driven by the oncogene KRAS, which is normally farnesylated. In cases where the farnesylation machinery is inhibited, however, KRAS eludes inactivation by using an alternative prenylation pathway in which the protein is geranylgeranylated. In order to study this alternative prenylation, large quantities of accurately processed protein are required...
June 21, 2018: Protein Expression and Purification
Andrew G Diamos, Hugh S Mason
Recombinant virus-like particles (VLPs) are proven to be safe and effective vaccine candidates. We have previously described a plant-based recombinant protein expression system based on agroinfiltration of a replicating vector derived from the geminivirus bean yellow dwarf virus (BeYDV). The system has been systematically optimized to improve expression and reduce cell death in Nicotiana benthamiana leaves. Using these modifications, we show that VLPs derived from genotype GII.4 norovirus, the leading cause of acute gastroenteritis worldwide, can be produced at >1 mg/g leaf fresh weight (LFW), over three times the highest level ever reported in plant-based systems...
June 14, 2018: Protein Expression and Purification
Xin Yong, Wenfeng Hu, Xue Zhou, Jing Wang, Ezra Burstein, Da Jia
The sorting nexin (SNX) family proteins play an essential role in vesicular transport, cell signaling, and membrane remodeling. The SNX members SNX1/2 and SNX5/6 form dimers, and mediate endosome-to-trans Golgi network (TGN) transport through coordinating cargo selection and membrane remodeling. It is well-known how a SNX-BAR protein forms a homodimer; however, it is less clear how a heterodimer is formed. Here a detailed expression and purification protocol of the SNX1/SNX6 complex, from both worm and human, is described...
June 14, 2018: Protein Expression and Purification
Hualong Xi, Jinfei Yu, Qing Sun, Jingcai Lu, Tiejun Gu, Xiaonan Guo, Bo Li, Xiaorui Chen, Kaixin Zhang, Wei Kong, Yongge Wu
Streptococcus pneumoniae is a major pathogen that causes life-threatening diseases, such as pneumonia, otitis media, bacteremia, and meningitis, worldwide and especially in young children and the elderly. Pneumococcal surface protein A (PspA) is a widely studied candidate protein vaccine that represents a promising replacement for current polysaccharide and polysaccharide-conjugate vaccines. In this study, we describe a simple method to produce PspA of clade 4 from an Escherichia coli expression system using hydroxylapatite and ion-exchange chromatography...
June 14, 2018: Protein Expression and Purification
B B Gengenbach, C R Müschen, J F Buyel
Cardiovascular diseases are a prevalent cause of morbidity and mortality especially in industrialized countries. The human phosphatase and actin regulator 1 (PHACTR1) may be involved in such diseases, but its precise regulatory function remains unclear due to the large number of potential interaction partners. The same phenomenon makes this protein difficult to express in mammalian cells, but it is also an intrinsically disordered protein that likely aggregates when expressed in bacteria due to the absence of chaperones...
June 13, 2018: Protein Expression and Purification
Xin Liu, Hai Wang, Bin Wang, Li Pan
Bacillus subtilis is widely used for the large-scale industrial production of proteins. In this study, the transcriptomes of B. subtilis 168 and B. megaterium DSM319 cells grown in stationary phase were analyzed to expand the repertoire of highly-active promoters for high-level protein expression based on the transcriptomes of these Bacillus strains. 24 genes with the highest expression levels among 2048 highly expressed gene families were chosen to examine promoter activity. The activities of four promoters with the beta-galactosidase (bgaB) gene as a reporter were stronger than those of the well-characterized strong promoter P43...
June 9, 2018: Protein Expression and Purification
Jan-G Vermeulen, Felicity Burt, Esta van Heerden, Errol Cason, Muriel Meiring
The development of therapeutic antibodies in their various forms has been a constant challenge since the development of the first monoclonal antibodies in 1975. This is especially true for the development of therapeutic single chain variable (scFv) fragments in Escherichia coli. In a previous study the selection of a tissue factor inhibiting single chain variable fragment (TFI-scFv) isolated from the Thomlinson I + J phage libraries was described. Although the initial findings were promising, additional characterization of the antibody fragment and subsequent application was hampered due low protein yield...
June 9, 2018: Protein Expression and Purification
Dujun Wang, Li Zhao, Jiayi Jiang, Jia Liu, Dan Wang, Xiaofeng Yu, Yuan Wei, Zhen Ouyang
1-Deoxynojirimycin (DNJ) is the main bioactive compound of Morus alba L.. DNJ has pharmacological effects, including blood sugar level regulation and antiviral activity. In this study, the mulberry lysine decarboxylase gene (MaLDC), which is involved in the biosynthesis of DNJ alkaloids, was cloned, expressed, and functionally verified. MaLDC was induced and expressed in Escherichia coli BL21 (DE3). The recombinant soluble MaLDC protein had a relative molecular mass of 24.0 kDa. The protein was purified by Ni-NTA separation...
June 9, 2018: Protein Expression and Purification
Weihua Jin, Shuai Li, Jiale Chen, Bing Liu, Jie Li, Xueliang Li, Fuming Zhang, Robert J Linhardt, Weihong Zhong
Heparan sulfate (HS), is a glycosaminoglycan (GAG) involved in various biological processes, including blood coagulation, wound healing and embryonic development. HS 3-O-sulfotransferases (3-OST), which transfer the sulfo group to the 3-hydroxyl group of certain glucosamine residues, is a key enzyme in the biosynthesis of a number of biologically important HS chains. The 3-OST-1 isoform is one of the 7 known 3-OST isoforms and is important for the biosynthesis of anticoagulant HS chains. In this study, we cloned 3-OST-1 from the rat brain by reverse transcription-polymerase chain reaction (RT-PCR)...
June 9, 2018: Protein Expression and Purification
Li Wang, Min Fan, Chao Zeng, Wei Li, Qikang Hu, Wenliang Liu, Xingchun Huang, Guiyuan Li, Fenglei Yu
TMEM8B-a protein is the longer, predominant isoform of the TMEM8B gene product, which is a tumor metastasis suppressor in nasopharyngeal carcinoma (NPC) and lung cancer. TMEM8B-a is rapidly degraded via the proteasome pathway mediated by ezrin in many NPC and lung cancer cell lines, but TMEM8B-a is not ubiquitinated. In this study, we report the recombinant production of full-length modified TMEM8B-a in mammalian cells. We used the PiggyBac transposon system to efficiently generate normal and lung cancer cell lines with stable TMEM8B-a protein expression...
June 8, 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...
June 7, 2018: Protein Expression and Purification
Yan Xie, Yan-Tao Yang, Wei Shi, Xia Ai, Xu-Guang Xi
AG1, a member of the DUF1220 protein family, exhibits the most extreme human lineage-specific copy number expansion of any protein-coding sequence in the genome. These variations in copy number have been linked to both brain evolution among primates and brain size in humans. Unfortunately, our current understanding of the structure and function of these proteins is limited because current cloning and expression techniques fail to consistently produce recombinant protein for in vitro studies. The present work describes a method for amino acid and DNA sequence optimization and synthesis, recombinant protein expression and analysis of two AG1 fragments, AG11-843 and AG11-1581 ...
June 2, 2018: Protein Expression and Purification
Juanjuan Yang, Yindi Liu, Shuli Xu, Haiying Lin, Chun Meng, Donghai Lin
The trans-translation system is recognized as an excellent target for developing new drugs to rapidly sterilize Mycobacterium tuberculosis (TB) infection and significantly shorten TB treatment duration. As a vital component of the trans-translation system for rescuing stalled ribosomes, the SmpB protein from Mycobacterium tuberculosis (MtbSmpB, 1-160 a. a.) mediates tmRNA binding to stalled ribosomes through forming a complex with tmRNA. So far, few works have been conducted to prepare, characterize biophysical properties and determine three-dimensional structure for the full-length MtbSmpB protein...
May 29, 2018: Protein Expression and Purification
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