Expression Vectors for Enzyme Restriction- and Ligation-Independent Cloning for Producing Recombinant His-Fusion Proteins

2008 ◽  
Vol 23 (3) ◽  
pp. 680-686 ◽  
Author(s):  
Blanca de las Rivas ◽  
José Antonio Curiel ◽  
José Miguel Mancheño ◽  
Rosario Muñoz
Keyword(s):  
Fusion Proteins ◽  
Expression Vectors ◽  
Ligation Independent Cloning

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

scholarly journals Preparation and Characterization of Anti-CeTNT-1 and Anti-CeTNT-4 Antibodies

1970 ◽  
Vol 44 (3) ◽  
pp. 371-376
Author(s):  
M Ziaul Amin ◽  
Hiroaki Kagawa ◽  
Mohammed A Satter
Keyword(s):  
Fusion Proteins ◽  
Tissue Specificity ◽  
Body Wall ◽  
Troponin T ◽  
Specific Interaction ◽  
The Body ◽  
Expression Vectors ◽  
Antibody Specificity ◽  
Sds Page

Among the four CeTNT isoforms, CeTNT-1, CeTNT-2 are body wall types, CeTNT-4 is pharynx type and CeTNT-3 is expressed in both the body wall and pharyngeal tissue. In our previous study, we used body wall and pharynx type anti-CeTNI, anti-CeTNC and anti-CeTM antibodies to observe the tissue specific interaction of the TNI isoforms with others TN subunits and tropomyosin isoforms. To extent the interaction study of CeTNT isoforms, in this study, we prepared and characterized the body wall type anti-CeTNT-1 and pharynx type anti- CeTNT-4 antibodies. For the preparation of the anti-CeTNT-1 and anti-CeTNT-4 antibodies, in this study we constructed the pCTNT-1 and pCTNT-4 expression vectors. The sub-cloned of the pCTNT-1 and pCTNT-4 expression vectors were verified by DNA sequencing. These expression vectors were used to generate fusion proteins of the body wall, TNT-1 and pharyngeal TNT-4 isoforms in Escherichia Ecoli. The expression of these fusion proteins were confirmed by SDS-PAGE analysis. The anti-CeTNT-1 and anti-CeTNT-4 antibodies were prepared in the rabbit by using the gel cut of the CeTNT-1 and CeTNT-4 fusion proteins. The antibody specificity of the CeTNT-1 and CeTNT-4 fusion proteins was also judged by Western-analysis using prepared anti-CeTNT-1 and anti-CeTNT-4 antibodies. The antibody specificity results indicated that anti-sera against each of both the body wall type TNT-1 and pharynx type TNT-4 isoforms had tissue specificity. Key words: Troponin T, Caenorhabditis elegans, Body wall, Pharynx DOI: 10.3329/bjsir.v44i3.4413 Bangladesh J. Sci. Ind. Res. 44(3), 371-376, 2009

scholarly journals Targeted regulation of plasmid DNA expression in eukaryotic cells with a methylated-DNA-binding activator

2021 ◽  
Author(s):  
Isioma Enwerem-Lackland ◽  
Eric Warga ◽  
Margaret Dugoni ◽  
Jacob Elmer ◽  
Karmella A. Haynes
Keyword(s):  
Dna Binding ◽  
Plasmid Dna ◽  
Fusion Proteins ◽  
Genomic Dna ◽  
Transcriptional Start Site ◽  
Eukaryotic Expression ◽  
Expression Vectors ◽  
Binding Region ◽  
Methylated Dna ◽  
Dependent Activity

Purpose: Targeted regulation of transfected extra-chromosomal plasmid DNA typically requires the integration of 9 - 20 bp docking sites into the plasmid. Here, we report an elegant approach, The Dpn Adaptor Linked Effector (DAL-E) system, to target fusion proteins to 6-methyladenosine in GATC, which appears frequently in popular eukaryotic expression vectors and is absent from endogenous genomic DNA. Methods: The DNA-binding region from the DpnI endonuclease binds 6-methyladenosine within the GATC motif. We used a Dpn-transcriptional activator (DPN7-TA) fusion to induce gene expression from transiently transfected pDNAs. Results: We validated methylation-dependent activity of DPN7-TA with a panel of target pDNAs. We observed stronger transactivation when GATC targets were located upstream of the transcriptional start site in the target pDNA. Conclusion: DAL-E, consisting of a 108 aa, 12 kD DNA-binding adaptor and a 4 bp recognition site, offers a genetically-tractable, tunable system that can potentially be redesigned to recruit a variety of regulators (e.g. activators, silencers, epigenome editors) to transfected plasmid DNA.

scholarly journals A Method for Rapid Screening, Expression, and Purification of Antimicrobial Peptides

2021 ◽  
Vol 9 (9) ◽  
pp. 1858
Author(s):  
Yingli Zhang ◽  
Zhongchen Li ◽  
Li Li ◽  
Ben Rao ◽  
Lixin Ma ◽  
...  
Keyword(s):  
Antimicrobial Peptides ◽  
Fusion Proteins ◽  
Expression System ◽  
Inhibition Zone ◽  
Eukaryotic Expression ◽  
Biologically Active ◽  
Rapid Screening ◽  
Expression Vectors ◽  
Expression And Purification ◽  
E Coli

In this study, a method for the rapid screening, expression and purification of antimicrobial peptides (AMPs) was developed. AMP genes were fused to a heat-resistant CL7 tag using the SLOPE method, and cloned into Escherichia coli and Pichia pastoris expression vectors. Twenty E. coli and ten P. pastoris expression vectors were constructed. Expression supernatants were heated, heteroproteins were removed, and fusion proteins were purified by nickel affinity (Ni-NTA) chromatography. Fusion proteins were digested on the column using human rhinovirus (HRV) 3C protease, and AMPs were released and further purified. Five AMPs (1, 2, 6, 13, 16) were purified using the E. coli expression system, and one AMP (13) was purified using the P. pastoris expression system. Inhibition zone and minimum inhibitory concentration (MIC) tests confirmed that one P. pastoris¬-derived and two E. coli-derived AMPs have the inhibition activity. The MIC of AMP 13 and 16 from E. coli was 24.2 μM, and the MIC of AMP 13 from P. pastoris was 8.1 μM. The combination of prokaryotic and eukaryotic expression systems expands the universality of the developed method, facilitating screening of a large number of biologically active AMPs, establishing an AMP library, and producing AMPs by industrialised biological methods.

Two Expression Vectors, Designated as pGEX-CDON and pC89S4-CDON, for Producing GST-CDON and pVIII-CDON Fusion Proteins

2013 ◽  
Vol 726-731 ◽  
pp. 505-510
Author(s):  
Fu Yong Xu ◽  
Ren Rong Liu ◽  
Ling Xu ◽  
Xue Mei Qiu ◽  
Li Xin Zhu
Keyword(s):  
Linear Equation ◽  
Fusion Proteins ◽  
Competitive Inhibition ◽  
Elisa Test ◽  
Linear Range ◽  
Expression Vectors ◽  
Random Peptide Library ◽  
Random Peptide ◽  
Inhibition Elisa ◽  
Better Than

Deoxynivalenol (DON) mimotope, designated as CDON, is an epitope (CMRPWLQ) immunoscreened from a phage-displayed random peptide library. In order to replace the conjugated toxin with non-toxic recombinant proteins in ELISA, two novel expression vectors, which were designated as plasmid pGEX-CDON and phagemid pC89S4-CDON for producing GST-CDON and pVIII-CDON fusion proteins in E.coli were constructed. After purification, both GST-CDON and pVIII-CDON fusion proteins show good reactogenicity with an anti-DON antibody in a competitive inhibition ELISA test. When GST-CDON was used as coating antigen, the linear range of the competitive inhibition ELISA is from 62ng/ml to 410ng/ml, the linear equation is Y= 186.6-23.87Ln (X), IC50 is 194ng/ml. For pVIII-CDON as coating protein, the linear range of the competitive inhibition ELISA is from 20ng/ml to 470ng/ml, the linear equation is Y = 161.3-25.49Ln (X), R2=0.9962, IC50 is 94ng/ml. ELISA analysis and comparison show the reactogenicity and specificity of pVIII-CDON binding to anti-DON antibody are better than GST-CDON fusion protein. The pVIII-CDON is promising in establishing an ELISA without the use of the toxic mycotoxin conjugate.

scholarly journals DNA Vaccines Expressing Different Forms of Simian Immunodeficiency Virus Antigens Decrease Viremia upon SIVmac251 Challenge

2005 ◽  
Vol 79 (13) ◽  
pp. 8480-8492 ◽  
Author(s):  
Margherita Rosati ◽  
Agneta von Gegerfelt ◽  
Patricia Roth ◽  
Candido Alicea ◽  
Antonio Valentin ◽  
...  
Keyword(s):  
Viral Load ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Dna Vaccines ◽  
Rhesus Macaques ◽  
Viral Proteins ◽  
Degradation Pathway ◽  
Expression Vectors ◽  
Native Proteins ◽  
Sivmac251 Challenge

ABSTRACT We have tested the efficacy of DNA immunization as a single vaccination modality for rhesus macaques followed by highly pathogenic SIVmac251 challenge. To further improve immunogenicity of the native proteins, we generated expression vectors producing fusion of the proteins Gag and Env to the secreted chemokine MCP3, targeting the viral proteins to the secretory pathway and to a β-catenin (CATE) peptide, targeting the viral proteins to the intracellular degradation pathway. Macaques immunized with vectors expressing the MCP3-tagged fusion proteins developed stronger antibody responses. Following mucosal challenge with pathogenic SIVmac251, the vaccinated animals showed a statistically significant decrease in viral load (P = 0.010). Interestingly, macaques immunized with a combination of vectors expressing three forms of antigens (native protein and MCP3 and CATE fusion proteins) showed the strongest decrease in viral load (P = 0.0059). Postchallenge enzyme-linked immunospot values for Gag and Env as well as gag-specific T-helper responses correlated with control of viremia. Our data show that the combinations of DNA vaccines producing native and modified forms of antigens elicit more balanced immune responses able to significantly reduce viremia for a long period (8 months) following pathogenic challenge with SIVmac251.

scholarly journals Mammalian Expression Vectors for Epitope Tag Fusion Proteins that Are Toxic in E. coli

BioTechniques ◽  
2002 ◽  
Vol 33 (6) ◽  
pp. 1218-1222 ◽  
Author(s):  
Rolf Jakobi ◽  
Corine C. McCarthy ◽  
Mark A. Koeppel
Keyword(s):  
Fusion Proteins ◽  
Expression Vectors ◽  
Epitope Tag ◽  
E Coli ◽  
Mammalian Expression
Sign in / Sign up

Export Citation Format

Share Document