This website uses cookies to ensure you have the best experience. Learn more

Expression And Purification Of Recombinant Linker Histone H1a21c Proteins From Escherichia Coli

722 words - 3 pages

Two restriction enzymes, Nde1 and Xho1, enabled the gene coding for the H1a21c protein to be successfully inserted into the pET22b plasmid. However, since the pET22b vector contains a His- tag sequence, it was important that the Xho1 enzyme to introduce a stop codon that prevented a His- tag to be attached to the H1 protein.
The SP FF ‘fast S’ column was used to purify the H1 protein based on the ionisation state of the protein. Referring to the structure of H1 proteins, they contain a high level of lysine residues (compared to core histones), thus making them relatively basic (Johns, 1971; Cole, 1984). The cysteine residue increased this positive nature of the H1 protein, allowing the ...view middle of the document...

This was a crucial step in order to purify the H1a21c protein further. Hydrphobic interaction chromatography promotes protein separation by interacting hydrophobic ligands and non- polar regions on the protein (Melander and Horva´th, 1977; Fausnaugh and Regnier, 1986; Roe, 1989). Hydrophobic interactions occurred between the H1 protein and the phenyl ligand on the phenyl sepharose column.
An initial high salt concentrated buffer enhanced the adsorption of the H1 protein by further exposing its hydrophobic residues (Melander and Horva´th, 1977; Fausnaugh and Regnier, 1986; Roe, 1989). In order to elute the H1 protein, a decreasing concentrated salt buffer was used in order to de- expose the hydrophobic residues, and this was confirmed by the decrease of conductance (Figure 5) as the proteins eluted.
A single defined peak (Figure 5) represented the elution of the H1 protein together with few contaminants. TCA precipitation was conducted in order to precipitate and concentrate the H1 protein for further SDS- PAGE analysis. It is important to note that the TCA precipitation method does not favour the precipitation of unfolded proteins (Rajalingam et al., 2009). The SDS- PAGE gel showed a more purified H1 protein, as seen by three distinct bands...

Find Another Essay On Expression and purification of recombinant Linker histone H1a21c proteins from Escherichia coli

Microbiology lab report on Escherichia coli and Staphylococcus aureus

825 words - 3 pages Microbiology Lab ReportPractica #1BTC307LAmber AmelingmeierThursday, September 18, 2008OBJECTIVESIn this lab experiment two different types of bacteria, Escherichia coli and Staphylococcus aureus, were grown singly and mixed on four different types of agar in order to observe the varying morphologies within the colonies. Resulting data was analyzed to provide understanding of the use of differing culture media and conditions for bacterial

Pros and Cons of Recombinant DNA Technology

1490 words - 6 pages : complementary DNA cloning and expression in bacteria." Science 205.4406 (1979): 602-607. 4. Martineau, Belinda. "First fruit: the creation of the Flavr Savr tomato and the birth of biotech foods." (2002). 5. Schnieke, Angelika E., et al. "Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts." Science 278.5346 (1997): 2130-2133. 6. Williams, Daniel C., et al. "Cytoplasmic inclusion bodies in Escherichia coli producing biosynthetic human insulin proteins." Science 215.4533 (1982): 687-689.

Purification of Milk Process and Chemical Reactions

2473 words - 10 pages alkaline phosphatase detection in dairy products using a modified rapid chemiluminescent method and official methods. J Food Prot 74:1144–54. doi: 10.4315/0362-028X.JFP-10-422 Atlan D, Portalier R (1987) Purification of extracellular alkaline phosphatase released by Escherichia coli excretory mutants. Appl Microbiol Biotechnol. doi: 10.1007/BF00256661 Chaudhuri G, Chatterjee S, Venu-Babu P, et al. (2013) Kinetic behaviour of calf intestinal alkaline

Integral Proteins are of Two Types: Transmembrane Proteins and Lipid Anchored Proteins

914 words - 4 pages proteins in the membranes, we can still determine two different types of integral proteins through part of the experiment. In this experiment, the integral membrane protein MaIF, which is required for maltose transport in Escherichia coli, was chosen to do the In-Gel Trypsin digestion experiment (Boyd et al 1987). Because MaIF integral protein contains many different types of proteins, a mixture of antibodies to alkaline phosphatase and &beta

An Account of Proteins and Their Structure

1183 words - 5 pages the protein is attached is called the prosthetic group. Egg yolk, haemoglobin and other pigments are all examples of conjugated proteins. If a protein is combined with a carbohydrate, the resulting compound is called a glycoprotein. Usually the protein forms the core of the molecule and the carbohydrate consists of a branched polysaccharide chain projecting from it. The function of a protein is directly related to

Bromination of Benzene Synthesis and Purification of Bromobenzene

559 words - 2 pages BROMINATION OF BENZENE SYNTHESIS AND PURIFICATION OF BROMOBENZENE: PROCEDURE DATA TABLE Chemical     Boiling point C     Melting Point C     Density g/mL     Solubility Benzene     80.1     5.5     0.88     Slightly in H2O Toluene&nbsp

Freedom of Speech and Expression

1257 words - 6 pages notion of autonomy (Rawls 1980, 554). Therefore restrictions on our freedom of expression correlate to restrictions on our autonomy. The doctrine of freedom of expression was written to single out the protected acts in which hold us to be immune from restrictions despite the existing facts that have consequences or harms us to be sufficient to the justifications and impositions of legal sanctions. Oliver Wendell Holmes believed that

Uniforms and Freedom of Expression

1256 words - 5 pages Does wearing a school uniform really make a difference in the learningprocess? Ever wonder what is the purpose of wearing a school uniform in thefirst place? If all of the students are dressed alike, it seems like it would bepretty tough to express your freedom of expression through attire. Manystudents, parents, and administrators have different opinions on the strictenforcement of a wearing school uniform. Some religious students might notbe

The Structure and Role of Proteins in Cell Membranes

1564 words - 6 pages permeable. It is also structural and keeps the cell contents together and separate from other cells. Furthermore it allows communication between cells and allows recognition of other external substances. Membranes that surround cell organelles are also very similar to the cell membrane. Membranes are composed of phospholipids, which form the bulk of the membrane, proteins, which are scattered around in the membrane. There may also

The Functions of Proteins in Plants and Animals

1083 words - 4 pages from sickle cell anaemia. Antibodies have a tertiary structure and are globular to ensure that the correct antigen attaches to it. If however, the shape of the binding site changes a mutation occurs and it no longer able to fight the same illness. Fibrogen is present in the blood plasma as part of the blood clotting mechanism of the body. Some proteins have a structural function such as collagen, elastin and keratin

Problem How is the presence of carbohydrates, proteins and fats

470 words - 2 pages Problem How is the presence of carbohydrates, proteins and fats identified in various foods? Background Information Organic Substances are mostly found in natural waters , for example: the aminoacids, carbohydrates, vitamins, humic acids, fulvic acid, lignins, pectins, enzymes and others.Inorganic substances are often found in matter compositions like in minerals.The difference between an organic substance and an inorganic substance is that

Similar Essays

Stable Expression And Purification Of Recombinant Extracellular Domain Of Her2 Protein

1748 words - 7 pages 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

Escherichia Coli Related Cystitis Prevalence And Pathogenicity

1519 words - 7 pages When one speaks of transmittable diseases one does not often think that cystitis is one of the most prevalent. Cystitis effects both genders, the young, the old and can lead to complications that can do irreparable damage to the urinary tract. The purpose of this paper is to discuss the disease of cystitis and relate the specific virulence factors of Escherichia coli to the prevalence and symptoms of cystitis. This will be accomplished by

Mechanisms Of Sex In Escherichia Coli

656 words - 3 pages The discovery of the gene transfer mechanisms could be attributed by the work of Lederberg and Tatum back in 1946. Using Escherichia coli(E.coli) as their model, they proposed the genetic materia of E.coli could be exhanged via sexual process. In order to prove their hypothesis, they mutated 2 wild type E.coli strains(K12) using X-ray or ultra-violet radiation to produce Y-10 and Y-24 mutant strains. The former was auxotrophic to threonine

Escherichia Coli Absorbancy Of Green Florescent Protein Through Heat Shock

1459 words - 6 pages GFP was derived from a complementary DNA of the Aequorea victoria. The protein expresses a fluorescent product when in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Other substrates or cofactors are not needed to fluoresce, so the GFP is quite versatile. It also can be used to monitor the gene expression or protein localization in living organisms without damage to the cells. (Chalfie 1995) When pGFPuv is expressed