cloning expression and purification of monoclonal antibodies in scfv fc format

... Fig Overexpression of HRT1 and HRT2 in E coli and purification of HRT2 The pETHRT1 and pETHRT2 were constructed and introduced into E coli BL21(DE3) The expression of these genes were induced ... pETHRT1 and pETHRT2 Each of the expression plasmids was used for transformation of E coli BL21(DE3), and mL of an overnight culture of the transformant in Luria–Bertani medium containing 50 lgÆmL)1 ... containing an 870 bp ORF, flanked by a 155 bp 5¢-UTR and a 254 bp 3¢-UTR including a poly(A) tail of 15 bp (Fig 1A) The cDNA of HRT2 was 1051 bp in length containing an 852 bp ORF (Fig 1B) and...

Ngày tải lên: 07/03/2014, 21:20

... that a subset of the Vps class E proteins initiate ubiquitin dependent vacuolar protein sorting by selectively binding ubiquitinated cargo and directing sorting of these cargoes into MVB vesicles ... protein-protein interactions and are generally found in cytoskeleton proteins and transcription factors Thus the structure of Osh1p and Osh2p is suggestive of being able to bind both a phosphoinositide ... of the similar genes and proteins in yeast and human and much of the work in defining the role of sterol in eukaryotic membranes has been done using the yeast model system In this project, we...

Ngày tải lên: 03/10/2015, 20:57

... folding of conotoxins is adding necessary folding information to the mature polypeptides For example, the C-terminal Gly that is used for amidation of the mature form of x-conotoxin containing ... protein has a calculated molecular mass of 54 913.7 Da and an isoelectric point of 4.6 The cPDI contains four thioredoxin domains and an acidic C-terminal tail (a, b, b¢, a¢ and c) Two thioredoxin ... conotoxins have been thoroughly investigated, and two distinct folding mechanisms have been unveiled [20] Through comparison of folding kinetics and thermodynamics, the folding mechanism of tx3a and...

Ngày tải lên: 07/03/2014, 05:20

... pathologies and because of the potential use of the toxins as biological weapons Alteration of their MHC and TCR binding capacity by site directed mutagenesis could be useful in the development of vaccines ... stocks of these clones were maintained at )70 °C TCR production and purification Luria–Bertani broth (LB) agar plates containing 50 lgÆmL)1 of kanamycin or 100 lgÆmL)1 of ampicillin were incubated ... superantigens (shown in cyan) Residue Cys26 is shown in red Equilibrium and kinetic parameters for SSA binding to different TCR b chains were determined in a resonance mirror using an IAsys instrument...

Ngày tải lên: 07/03/2014, 16:20

... of Monitoring and Management of Crop Diseases and Pest Insects, Ministry of Agriculture, Nanjing, China Table Oligo DNA primers used in the present study Restriction sites or mutation sites in ... Ib Expression and purification of AATB3 and its mutants To produce recombinant AATB3 and the three mutant proteins, the aatB3 gene and its mutants were expressed in E coli The recombinant proteins ... Glu and Asp, and the formation of Glu and Asp is used to synthesize Gln and Asn, respectively These four residues are common components in lipopeptides, such as surfactin, iturin and fengycin...

Ngày tải lên: 14/03/2014, 23:20

... describe the cloning and expression of the M tuberculosis tps gene in E coli, and the production of active TPS in these cells The properties of the recombinant enzyme have been determined, and are ... by cutting a strip of the paper into 0.5 cm pieces, from the origin to the solvent front, and counting each strip in a scintillation counter RESULTS Cloning and expression of M tuberculosis TPS ... electroporated into the E coli strain HMS-F [29] The resulting ampicillin- and tetracycline-resistant transformant was cultured with and without IPTG, and expression of a (His)6-tagged protein of the...

Ngày tải lên: 17/03/2014, 10:20

... Albert Einstein College of Medicine, New York The E coli strains TOP10 and BL21Star (DE3) (Invitrogen) were used for cloning and expression studies, respectively E coli strains were cultured in Luria–Bertani ... Q-TOF MS, and identified in the M smegmatis genome (shown in bold type and underlined) These peptides allowed the gene for TreS to be identified in the genome and its cloning and expression in ... activity of TreS was routinely measured by determining the formation of reducing sugar when enzyme was incubated with trehalose Assays were carried out in a final volume of 100 lL, containing 40...

Ngày tải lên: 23/03/2014, 13:20

... 30 °C in Luria–Bertani (LB) broth containing 0.5% glucose and 60 lgÆmL)1 kanamycin Thirty milliliters of culture was used to inoculate L of M9 containing 0.5% glucose and 60 lgÆmL)1 kanamycin These ... using this signal sequence and it has a convenient NotI site at its C terminus, which allows cloning in- frame with alanine as the first amino acid of the mature protein An alignment of the amino ... mass 94 705 Da), indicating that the signal peptide was being cut in different places, resulting in the addition of up to 17 extra amino acids at the N terminus of the protein It is not clear...

Ngày tải lên: 23/03/2014, 21:20

... protein standards (C) Protein bands after affinity chromatography and renaturing process Lanes and 2, protein bands after separation by affinity column; lanes and 4, protein bands after renaturing ... by inhibiting the synthesis of one of their cell wall layers [40,41] Cathelicidins can kill both Grampositive and -negative bacteria, enveloped viruses including HIV, and fungi including Candida ... N-terminus Noticeably, four cysteines that are conserved in the cathelin domain of all cathelicidins identified to date are also invariantly spaced in Cc-CATHs precursor [11] (Fig 3) The processing...

Ngày tải lên: 28/03/2014, 23:20

... was determined by detecting the formation of nitrite using the method of Ida et al [31] Km and Vmax of purified NaoA were determined from a double-reciprocal plot according to the LineweaverBurk ... The pI of NaoA was determined from a standard curve of pI and migration distance (cm) of protein standards Enzyme assay and analytical methods E coli BL21(DE3) containing plasmid pNA101 was grown ... fragments of m/z 58 (M+) and m/z 43 consistent with those of acetone standard Properties of NaoA The pI of NaoA is about 5.2 according to the standard plot between the protein’s pI and its migration...

Ngày tải lên: 31/03/2014, 08:20

... Figure strategy for expression of LASV proteins GP1, GP2, and NP in E coli using pMAL vectors Cloning Cloning strategy for expression of LASV proteins GP1, GP2, and NP in E coli using pMAL vectors ... strain and the pMAL-c2x vector background resulted in improved expression of both LASV glycoproteins, allowing us to achieve the highest yield of recombinant protein per gram of cell mass in an ... N-terminus Thus, recombinant LASV proteins are expressed in these systems with the correct N-termini, and in the case of NP and GP2, the two major truncated forms fall short of reaching the C-terminus...

Ngày tải lên: 20/06/2014, 01:20

... different organs, including the lungs [8,14] Our data may explain this rapid and efficient dissemination of SARS-CoV By slowing down expression of IFNs and their antiviral genes in the infected tissue ... Figure Interleukin production Interleukin production RNA samples of Caco-2 cells (A) and HEK 293 cells (B) described in Fig were investigated by RTPCR for the presence of IL-6 and IL-8 mRNAs by infected ... antiviral cytokine response of infected cells to a large extent Interestingly, however, induction of the chemokines IP-10 and IL-8 escaped suppression by SARS-CoV in Caco-2 cells, but not in HEK 293s...

Ngày tải lên: 20/06/2014, 01:20

... given in units per milligram (U/mg) protein One unit is defined as the amount of enzyme that catalyzes the oxidation of μmol of substrate per minute Results Cloning, expression and purification of ... sites of restriction endonucleases NdeI and XhoI for cloning with the BCA-kit and a standard curve of BSA in the same buffer in a range of 2-0.005 mg/mL was used Samples were measured in triplicates ... FMN/NADH-containing type II BVMOs were investigated up to now A reason might be the challenging overexpression of these enzymes in a heterologous host, since in contrast to type I BVMOs the oxygenating and...

Ngày tải lên: 21/06/2014, 02:20

... Yujin Kim et al expression of inhibin isotypes might be observed in seminiferous tubules in mice during testicular development This study examined the level and cellular localization of inhibin ... photographs of Western blots for inhibin isotypes α, βA and βB and beta-actin (A) Arrowheads indicate the positions of the inhibin isotypes (40∼47 kDa) and beta-actin (45 kDa) Minor bands at various ... Temporal expression pattern of the inhibin isotypes α, βA and βB during the postnatal development of mouse testis The protein levels of the inhibin α, βA and βB isotypes in the testes during postnatal...

Ngày tải lên: 07/08/2014, 23:22

... is a dynamic change in the levels of miRNAs within the same cell type but in different activation state Combined analyses using direct cloning of miRNAs, microarray profiling and RT-PCR, was used ... In silico bioinformatics analyses has helped a lot in predicting the potential targets of miRNAs Elucidating miRNA-target genes and their mechanism of action has given valid clues in understanding ... which miR-155 expression is controlled and finding its targets will bring more understanding of the mechanisms of tumor induction by miR-155 76 miR-181 miR-181 is highly expressed in the thymus...

Ngày tải lên: 08/08/2014, 17:20

... to the induction of c-jun and c-fos and AP-1-dependent transcription of MMP genes [10] In addition, triggering of the ceramide pathway in human keratinocytes results in overexpression of MMP-9 ... CO2, 95% air, in ml of DMEM supplemented with 10% fetal calf serum (FCS) DMEM containing FCS was removed and explants were incubated overnight in serumfree DMEM with insulin–transferrin–sodium selenite ... The amount of sGAG remaining within the cartilage explant after 24 hours of treatment with TNF-α and C2-ceramide in the presence and absence of 2-AP was determined by papain digestion and DMMB...

Ngày tải lên: 09/08/2014, 01:23

... level of PPP2R5C in K562 (CML cell line), and lower expression level in B-cell lines (Raji and Daudi) Mutation, deletion, or abnormal expression of tumorsuppressor genes is frequently identified in ... Unlike most Figure Expression level of PPP2R5C in human leukemic cell line A: T-cell lines and healthy CD3+ T cells, B: Myeloid cell lines and B cell lines and PBMCs from healthy individuals leukemia ... YQL, HTZ and YC coordinated the study and helped to draft the manuscript All authors read and approved the final manuscript Competing interests No potential conflicts of interest and financial...

Ngày tải lên: 10/08/2014, 21:23

... specific to it in DPV-infected cells We examined the expression of gE protein in DPV-infected cells using Western blotting, and analyzed the DPV gE gene transcription in DPV-infected cells using the ... Tyrosine-containing sorting motifs in regulating the intracellular traffic of membrane proteins [33,35] The Tyrosine-containing sorting motifs usually consist of a tetrapeptide bearing the sequence ... positive recombinant plasmid and used for protein expression Expression and Purification of the recombinant protein Expression of this fusion protein was regulated by an IPTG-inducible lac operator...

Ngày tải lên: 12/08/2014, 04:20

... tyrosine kinase) which is involved in BCR signaling, but also in the regulation of integrin activation [44] Moreover, recent work indicates that Syk is involved in CXCL12- and a4b1 integrin-induced ... effect of epratuzumab on the expression of the b1 integrin on the surface of B-cells In this regard, integrin complexes are composed of b and a subunits and form unique molecules, and b7 integrin ... T-cells since high expression of a4b1 integrin resulted in a loss of a4b7 integrin With this in mind, subsequent studies were performed to analyze whether down-modulation of b7 integrin on the...

Ngày tải lên: 12/08/2014, 15:21

... 3.6 Expression and purification of recombinant HSP20 (rHSP20) 64 3.6.1 Induced expression of recombinant protein (rHSP20) 64 3.6.2 Purification of rHSP20 by Affinity chromatography 65 3.7 Raising ... for maintaining the normal functions of H pylori proteins, assisting H pylori in combating against stress and survival in the stomach (Kamiya et al., 1998) 1.4 Heat shock proteins (HSPs) of Helicobacter ... conserved and widely expressed in both eukaryotes and prokaryotes that are detected in folding, transporting and stabilization of proteins in cells As one of the virulence factors, HSPs are indispensable...

Ngày tải lên: 12/09/2015, 10:19