3.1. Construction and expression of HER2 ECD
The gene encoding the human HER2 ECD (1968 bp in length) was amplified from cDNA synthesized on mRNA template. The PCR product was cloned into pcDNA3-SV5-BAP and pcDNA3-hεCH4-GPI vectors, downstream of the secretory signal sequences. Both recombinant vectors were verified by digestion and agarose gel size. Results of sequence analysis confirmed the sequences of following proteins HER2 ECD-SV5-BAP from pcDNA3 HER2 ECD-SV5-BAP, and HER2 ECD-hεCH4-GPI from pcDNA3 HER2 ECD-hεCH4-GPI recombinant vectors. For in vivo biotinylation of HER2 ECD, equimolar amounts of both plasmids pcDNA3 HER2 ECD-SV5-BAP and pCDNA3-BirA encoding the tagged HER2 ECD and BirA enzyme respectively, were transiently co-transfected into HEK293 cells. Biotinylation of ECD-HER2 was achieved in BAP sequence that codes 15 aa in C-terminus of SV5 tag by BirA biotin ligase. This is illustrated in Figure 1. Then, stable cell lines of HEK293 harboring the DNA encoding HER2 ECD-BAP and BirA were established. Clonal cells were expanded from Genticin resistant colonies. Different clones were analyzed with ELISA and Western Blotting analysis (Fig. 2).
3.2. Western blotting and ELISA analysis of the biotinylated HER2 ECD
Eight stably transfected clones were screened in Western blot analysis that 6 of them exhibited expression of HER2 ECD (Fig. 2-A). The recombinant protein in the cultural supernatant of these clones was analyzed by ELISA for their ability to bind streptavidin. All the six HER2 ECD-expressing clones showed the biotinylation of HER2 ECD in ELISA assay (Fig. 2-B). The medium of one selected clonal cells cultured in the presence of biotin, were dialyzed extensively against phosphate buffer to eliminate free biotin an...
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...tal efficacy [34]. Glycosylphosphatidylinositol (GPI) anchors play important roles in the surface expression of eukaryotic membrane proteins [35]. GPI precursor is synthesized in the endoplasmic reticulum and then is covalently attached as a posttranslational modification to the C-terminal of a protein that express a GPI attachment signal sequence [36]. This procedure (GPI-anchor attachment) leads to stable association of the target proteins with the membrane [16, 36]. We produced a cell line that stably express ECD HER2-GPI anchored protein on cell surface. Flow cytometry and Immunoflourescent microscopy assays showed that the cell surface expression of HER2 ECD is remarkable. This cell line can frequently be used in biophysical and structural studies of binding anti HER2 agents. Moreover, it is benefit to screening studies for the design of effective therapeutics.
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
Overall, as the concentration of the substrate increases, the enzyme activity increases up to a 70% of solution, where the enzyme activity starts to level off. The curve is polynomial because of the fact that the enzyme activity exponentially increases as the concentration of substrate increase; additional evidence for this is the fact that the gradient graph is constantly changing. The polynomial curve is shown because until 70% (the saturation point); this is because there are more casein substrate molecules that can successfully collide with the renin enzyme molecule, therefore increasing the rate of reaction.
...stem: roles of eclosion hormone and ecdysis triggering hormone. Journal of Experimental Biology, Vol. 200, pp. 869-881
...It allowed access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analysis. First, the pEGFP-N1 plasmid nucleotide sequence was found by using the NCBI nucleotide database program. SnapGene viewer illustrated the restriction enzyme cut sites used to cut EGFP gene from the pEGFP-N1 source plasmid. Then the pET-41a (+) vector sequence was found by using the AddGene Vector Database. A new DNA file representing the recombinant pET-41a (+)-EGFP plasmid was built by virtually cloning the EGFP gene insert into the pET-41a (+) vector sequence. The plasmid was virtually cut utilizing the pAD1 sense primer and pAD1 anti primer from the PCR procedure. A restriction digest experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cut the PCR product at least once was HgaI and XspI.
Receptor tyrosine kinase is a cell membrane receptor system that can trigger multiple cellular responses simultaneously. It requires two receptor tyrosine kinase proteins, which are initially individual polypeptides that each have a signal-binding site, an α helix spanning the cell membrane, and a tail of multiple tyrosines. When signal molecules bind to both proteins they attach through a process called dimerization, forming a dimer. This process activates, or phosphorylates, the ends of the tyrosines, also known as tyrosine-kinase regions. Once the dimer is activated, multiple inactive relay proteins are able to bind to the tyrosine-kinase regions. Each of these proteins trigger a cellul...
CP consists of a single domain with high α-helical content [4]. The N-terminal part this domain is surface exposed whereas the C-terminal region buried in the virion. Several experiments indicate the CP is an O-glycoprotein. Equal amounts of galactose and fructose residues are O-linked to an acetylated serine residue at the N-terminal region [2]. This mediates the formation of a structured...
Once binding has occurred, a cascade of signalling reactions will initiate, with Rho guanosine-5'-triphosphate (Rho GTPases) such as rho-asso...
Schulman, Joshua M., and David E. Fisher. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 28 Aug. 0005. Web. 24 Apr. 2014.
In this experiment, we perform a gel electrophoresis on the DNA. In this process, the enzymes were run through the gel electrophoresis to determine their relative sizes for each of them. The results of the certain DNA fragments are used in the final step, which is to construct a map of the DNA molecule. If we use different enzymes to cut DNA, then not every restriction site will be cut by all of the enzymes. The objective of this lab is to perform restriction enzyme of digesting plasmid DNA and constructing a map of plasmid from the results made from the experiment. Using this technique we understand what a DNA restriction enzyme is and how it works. In this process, the enzymes were run through the gel electrophoresis to determine their relative sizes for each of them. By following the experiment, we determined the positions of the restriction
Gene therapy works by introducing new and functioning genetic material to damaged genes to help it function and to produce beneficial proteins. If a gene is inserted directly into a cell, it usually will not function. So to complete this task, a vector, a modified virus is used to carry and deliver the new gene. There are two different categories of vectors than can be utilized in this process; recomb...
Shi, Y., & Zou, M. (2008). Progress in gene therapy research. In J. L. Lewis (ED.), Gene therapy and cancer research progress (pp. 23-130). New York, NY: Nova Science Publishers, Inc.
Distinct characteristics are not only an end result of the DNA sequence but also of the cell’s internal system of expression orchestrated by different proteins and RNAs present at a given time. DNA encodes for many possible characteristics, but different types of RNA aided by specialized proteins sometimes with external signals express the needed genes. Control of gene expression is of vital importance for an eukaryote’s survival such as the ability of switching genes on/off in accordance with the changes in the environment (Campbell and Reece, 2008). Of a cell’s entire genome, only 15% will be expressed, and in multicellular organisms the genes active will vary according to their specialization. (Fletcher, Ivor & Winter, 2007).
There are many functions lipids have. One of the main functions lipids are structural components in the cell. Lipids make up approximately 50% of the mass of most cell membranes. The lipids that are found in the cell membrane are called phospholipid. Phospholipid are the predominant lipids of cell membrane. Phospholipids aggregate or self-assemble when mixed with water, but in a different manner than the soaps and detergents. Because of the two pendant alkyl chains in phospholipids and the unusual mixed charges in their head groups, micelle formation is unfavorable relative to a bilayer structure.
The DENV envelope protein E, which is found on the virus surface, has a role as a mediating factor in the initial attachment of the virus to the host cell. Further, several cellular proteins and carbohydrate molecules that act as attachment factors interacting with the viral envelope protein E have been identified. These factors allow the virus population to concentrate on the cell surface thus increasing their chance of access to their target cellular receptor(s). Some of these known molecules that interact with the vi...