Essay On Proteolytic Degradation Tagss

This lab investigated the central dogma of molecular biology by studying the expression of jellyfish Green Fluorescent Protein in E. coli (Mardigan, 2011). After transforming E. coli with the GFP gene under the arabinose operon promoter, GFP expression was analyzed with and without arabinose. Induction was seen only in media with arabinose. To further study GFP protein and structure, an SDS-Page or gel electrophoresis was conducted where proteins were charged to allow them to move in an electrical field in terms of their size. To further study the role of the arabinose operon in GFP expression an overnight culture was done where the plate was frozen and incubated to induce optimal protein folding. The GFP was then purified. Centrifugation, use of buffers, and a column was used to separate the GFP and bacterial cells.

In Figure 3, there is a collected data with the total washes and the elution fraction with the RFUs from the Ni+2 Agarose column. E3 had the highest elution fraction with 8554 RFUs. In the Bradford assay, E3 had the highest protein amount with 65.5ug. The specific activity for E3 was about 1.28E5 RFUs/mg. Overall, in the Figure 3, the E1-E6 had most of the rGFPs due to being bonded in Ni+2. W1-W6 rGFP is much smaller than the E1-E6 amounts.

I did not get conclusive data from the gel I made. As you can see in figure 1, the bands that showed up on the gel were too cluttered to be able to measure them. So I could not compare protein bands between the fish species based on our gel. Instead, I used a default gel picture that another group did in the class to get my data. From their gel I was able to compare the different species. Table 1 shows the number of bands that were similar between the different fish species when they were compared. I was able to determine that fish species C (Tuna)

Second, in order to further confirm the information about characteristics and function of the targeting protein that we have

The genome of E. coli 0157:H7 was recently sequenced and contains 1,387 new genes absent in non pathogenic strains. Analysis of the genome suggests that there are a variety of potential virulence genes that have yet to be explored, including fimbrial and other adhesions, secretion systems, and toxins. Many of the virulence genes are similar to those associated with pathogenesis in Salmonella and Shigella. Most of them are found on pathogenicity islands from viral DNA that is transmitted to the bacteria through the activity of a lysogenic phage. The two most important toxins, Stx1 and Stx2 (for shiga-toxin) are found on such islands and are composed of 5 B subunits surounding an active A component, which is transported into the cell after the

coli BL21(DE3)pLysS cells and a single colony in a transform agar plate was inoculated into LB broth containing 50µg/ml kanamycin. When absorbance reading at 600 nm reached approximately 0.5, the expression of His-tagged Δ gD1-Δ gD2 protein was induced by the addition of 0.5 mM isopropyl-1-thio-β-D-galactoside (IPTG) at 20˚C overnight. The recombinant protein was purified by affinity chromatography as described previously.(12) The concentration of purified recombinant protein was measured by BCA assay according to the manufacturer’s instruction. The level of endotoxin in each purified protein was quantified as previously

Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

The newly formed ookinete survives in the harsh protease rich environment of the midgut due to protease resistant covering (Gass and Yeates, 1979). Carter and Kumar (1985) observed that ookinete surface proteins are responsible for protecting ookinetes from protease enzymes attack (Carter et al., 1988). These and other parasite surface proteins are also candidates for an antizygote-ookinete transmission-blocking

Protease, an enzyme that degrades proteins, then cuts of a small piece of the Protective Antigen. The leftover part of the Protective Antigen then forms into a ring-like structure with six other Protective Antigen-receptor pairs. [4] This creates sites where the Lethal Factor and Edema Factor can attach to the top side of the Protective Antigen. It then deludes the cell, and enters the cell through endocytosis. This creates an endosome with a bubble-like compartment surrounded by a membrane with the toxin-receptor inside. When this moves closer to the Lethal Factor and Edema Factor, it forms a barrel-like pore through the membrane. This is where the toxic Lethal Factor and Edema Factor will be released from to attack targets within the cell.

Proteases are dangerous to the cells, being inactive is the safe way for cells to handle the enzymes. They activate when they reached the stomach to help with the

During the short burst phase the catalytic triad, made up of Asp102, His57 and Ser195, is acylated, allowing cleavage of the peptide bond. Deacetylation occurs in the steady-state phase, which returns the enzyme to its original state, allowing for further enzyme-substrate complexes to form. These stages were identified by using a substrate analog, n-acetyl-phenylalanine p-nitrophenyl. A product

The processing of APP involves three proteases, α-, β-, and γ-secretases and two processing pathways, the amyloidogenic pathway and the anti-amyloidogenic pathway as shown in Figure 2 (14, 23). The Aβ is generated in amyloidogenic pathway which is mediated by β-, and γ-secretases (14). The β- secretase first sheds the ectodomain of APP and generates the APP carboxy-terminal fragment (βCTF) (14). Then the γ-secretase cuts at the transmembrane region of βCTF and releases the Aβ into the extracellular fluids like cerebrospinal fluid or plasma (14, 24). In the anti-amyloidogenic pathway α-secretase first cuts at the middle region of Aβ so a trauncated APP CTF (αCTF) is generated and after the transmembrane cut of γ-secretase, a truncated Aβ peptide is released (p3) (Figure 2; 25). The amyloidogenic and anti-amyloidogenic competes with each other (26-27). In addition, when the γ-secretase cuts at the transmembrane region of APP, the cut site is not restricted to a single position. The γ-secretase first cuts at

Proteases in the MEROPS protease database have been subdivided into families and clans on the basis of evolutionary relationships (http://merops.sanger.ac.uk) (Rawlings et al., 2014). A protease clan refers to proteases derived from a single common ancestor, and clans are subdivided into families. A protease family refers to a sub-group of proteases that share sequence similarity, either throughout the entire protein sequence or only within the catalytic domain. The Arabidopsis thaliana genome encodes 879 known and putative proteases, corresponding to approximately 3.2% of all Arabidopsis protein-coding genes (The Arabidopsis Information Resource). These proteases are distributed over 60 families that belong to around 30 different clans

Reports indicate that the extracellular serine protease inhibitor, C1INH, can effectively bind HABP2 in the blood (27). We therefore examined whether C1INH can colocalize with HABP2 in

A single colony of freshly grown E. coli was cultured overnight in 2YT before an amount moved to fresh 2YT to generate 20 mL at OD 0.5. A 1 mL 0 hr non-induced sample was taken before the addition of 1mM IPTG. 1 mL samples of OD 0.5 were then taken at (post–induction) 1 hr, 2 hr and 3 hr time-points for analysis through Western Blot.