Expression and Purification of Recombinant Linker Histone H1a21c Proteins from Escherichia Coli

This experiment was conducted as per the BCHM 310 Laboratory Manual [3]. The first objective of this experiment was to analyze the purity of the invertase fractions collected during experiment 6, and to determine the molecular weight of LDH-H4, LDH-M4 and invertase subunits. This was accomplished using sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, SDS, a negatively charged amphipathic molecule, was used to denature the proteins and to give each protein a similar charge-to-mass ratio [4]. As a result, most oligomeric proteins separated into individual subunits, and each subunit assumed a rod-like shape [4]. The distance travelled by each subunit, along the polyacrylamide gel, was a function of its molecular weight; where proteins with a greater molecular weight moved a smaller distance than proteins with lower weights [5]. Since SDS is not a reducing agent, and no other reducing agent was added, oligomers with disulfide bonds between subunits would have remained intact [4]. However, this was not expected to be problematic for analyzing invertase or LDH isozymes, as these proteins lack disulfide interactions between their subunits [2,6]. In addition, since invertase and LDH are homo-oligomers, each protein’s subunits were expected to migrate the same distance [2,6].

The absorption of proteins as well as thrombus formation on the pH sensors has been known

This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal

The purpose of this experiment was to separate proteins on the basis of their net charge at a particular pH. In cation exchange chromatography positively charged molecules are attracted to a negatively charged column. Conversely, in anion exchange chromatography, negatively charged molecules are attracted to a positively charged column. Experimental results could be monitored in a predictable way by controlling running pH, salt concentration, and by selecting the type of ion exchanger.

To mutagenize human Kif5A (WT), we performed PCR-mediated site-directed mutagenesis to insert our mutation into the plasmid vector (shown to the right). Next, we performed a restriction enzyme digest with Dpn1, to digest the parental plasmid.  The presence of the PCR product was confirmed by performing gel electrophoresis where the expected band of 6.8 kilobases was obtained. The remaining DNA was used in a bacterial transformation of DH5α to grow colonies. Liquid cultures were grown from overnight colonies and Minipreps were performed to purify the plasmid DNA. These plasmids were sent for sequencing, where the P278L mutation was confirmed.

The experiment was conducted in two main parts: in the first part, the single and double restriction digests were run to cut the plasmids using combinations of three different enzymes. In the second part, the fragments resulting from each reaction were separated using gel electrophoresis. 1) Restriction Digests In this part of the experiment, seven different samples were prepared with different combination of reactions. Given the unknown plasmid and three restriction enzyme (HindIII, BglI and PvuII), seven different Eppendorf tubes were prepared to account for 3 single digests, one with each of the three restriction enzymes, 3 double digests combining two enzymes at a time and alternating between all the possible combinations, and also for the control reaction where no enzymes were added to the plasmid.

For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2

Four more single restriction digest reactions were performed in the same manner described above. The four digest reactions were run with restriction enzyme EcoRI using the same components, concentration, and volume used in the master mix previously mentioned. Each reaction contained 8 μL of master mix and 2 μL of plasmids. The first two reaction were pKan and pBAG plasmids digested with EcoRI separately as mentioned above. The other two reactions were identical and were composed of recombinant plasmids obtained from second miniprep digested with EcoRI restriction enzyme.

Neufeld is able to determine both relative and quantified information about the concentration of a protein in various locations in a cell. One technique Neufeld uses frequently is the Western Blot, which employs the concept of gel electrophoresis. After choosing the group of cells she wants to examine, Neufeld treats the cells with a solution of sodium dodecyl sulfate (SDS), which causes the cells to lyse and the proteins to denature. Sodium dodecyl sulfate functions by disrupting the non-covalent interactions between molecules. Because proteins typically exhibit hydrogen bonding, hydrophobic interactions, and other intermolecular forces, their structures are greatly compromised. SDS is composed of a hydrophobic hydrocarbon tail and an ionic sulfate group. When SDS encounters a protein, the hydrocarbon tail dissolves the hydrophobic regions of the protein and the ionic sulfate group disrupts non-covalent ionic bonds. Furthermore, SDS causes the protein to hold a negative charge. To make sure the proteins are completely denatured, they are often boiled. Once the proteins are undoubtedly denatured, they are inserted into an acrylamide gel, through which an electrical current is run. The proteins, attracted to the anode due to their negative charge, migrate through small pores in the gel. Because movement through the pores is faster when the protein is small, the various proteins are essentially resolved according to their sizes; smaller

The transport activity is expressed as nmoles of substrate transported during the incubation time per milligram of the reconstituted protein and it is calculated with the following formula:

Two Polymerase Chain Reactions (PCR) were set up, one was digested with the restriction enzyme and the other served as the control. 11.0 µl of the forward primer solution, 11.0 µl of the reverse primer solution, and 3.0 µl of the

The pellet was resuspended in PBS containing 1% Triton X-100, and the solubilized proteins were designated as the membrane fractions.

Protein molecules are made soluble in buffered liquid and isotopically labeled. Therefore, allows thermodynamics, studies of the kinetics aspects of structures and interactions with other components.

The unique and primary proteins of DNA, the highly basic histone proteins have been found to be ubiquitous and conserved among eukaryotes. DNA and histones co-exist as a closely associated and highly coiled structure. The purpose of such a structural organization is to compact DNA; which would otherwise be too long to be accommodated within the restricted boundaries of a single cell. Core histones (H2A, H2B, H3 and H4) and Linker histones (H1) are the two types that this unique protein is composed of. These Core histone exhibit a structured organization with H2A and H2B forming dimers and H3 and H4 forming tetramers. Along with approximately 146 base pairs of DNA, these core histones form a heterotypic nucleosome complex; weighing about 206

one of the first proteins to be purified to the point where its molecular weight