Histamine-binding proteins (HBP) are ubiquitous biomolecules with pharmacologic importance, since they have an immunosuppressing activity, which can help prevent adverse health conditions such as histamine shock. …
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To facilitate further studies regarding the structure and function of HBP, there should be an efficient means by which HBP can be made available. For this, Pichia pastoris expression system was assessed for its competence in producing recombinant HBP. This was chosen for its effectiveness in glycosylating recombinant proteins. On the other hand, the HBP gene sequence used for this particular study was from cattle tick, Rhipicephalus microplus, which is considered to be an agricultural pest. If found to have therapeutic effect, the despised insect will be given a newly-discovered purpose. Culture and induction of transformed P. pastoris was able to produce c-myc epitope-containing proteins, potentially containing TC11485, as detected through dot blot and Western blot analysis. Future researches involve purification and characterization of the recombinant TC11485. Introduction Lipocalins 1. The structure-function relation in lipocalins Lipocalins are monomeric globular proteins comprised of a single polypeptide with 150-200 amino acid residues, and ubiquitous in all life forms. In fact, these proteins are abundant in plasma, tissue and secretory fluids of humans. Despite its weak sequence homology, they are characterized by a tertiary structural level of a conserved ?-barrel configuration with an amino-and carboxy-terminal ?-helix attachment, contributing to their similarities in function, which will be discussed in the later sections. The barrel is shaped like a cone, in which the tip is a hydrophobic core that protects the parcel, and the base open to solvent acts as an entry point into the cavity. In fact, the term lipocalin is derived from ‘calyx’, which is the Greek and Latin word for drinking vessel (Cheng, 2010; Schlehuber and Skerra, 2005). Classification of lipocalins is based on variations in the length of the terminal segments. Aside from the highly conserved tertiary structure, lipocalins also exhibit similar arrangement of exons and introns in their genes’ coding sequences (Cheng, 2010). Understandably, each lipocalin has a distinct amino acid sequence. For human lipocalins, a single unpaired cysteine (Cys) residue allows intermolecular covalent binding of a lipocalin to another protein. Apolipoprotein D (ApoD) binds with apolipoprotein A-II, and NGAL associates with matrix metalloproteinase IX Other than being differentiated based on amino acid sequence, lipocalins vary in the shapes that their structures can assume. Logically, capability for such changes influences the function of the protein. For example, neutrophil gelatinase-associated lipocalin (NGAL) opens more widely to become more funnel-like, while the mouse major urinary protein (MUP) closes the opening of the barrel to totally encapsulate the ligand (Schlehuber and Skerra, 2005). 2. Physiologic role of lipocalins This family of proteins primarily functions to transport or store compounds that are insoluble or chemically sensitive. Among the compounds transported by lipocalins are hydrophobic vitamins, pheromones, bilins, retinoids, lipids and steroid hormones, play significantly in transcription, enzymatic reactions and metabolism (Schlehuber and Skerra, 2005; Cheng, 2010). They deliver their ligands to the cell membrane receptor or to the targets (such as DNA) themselves. For example, the human plasma retinol-binding protein (RBP), the first lipocalin structurally characterized, transports the insoluble and highly oxidative vitamin A from the stores in the liver to the target tissues . ApoD transports progesterone and arachidonic acid, while NGAL has Fe(III)-enterobactin as its ligand (Schlehuber and Skerra, 2005). 3. Medical significance of lipocalins Because of their
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Bradford assays are among the methods used to determine the concentration of protein, relative to a standard. This technique is based on the formation of a complex between the dye, Brilliant Blue G and proteins in solution. The complex being formed causes a shift in the absorption peak from 465nm to 595nm.
Fig 1. Fluorescence intensities of a) tyrosine b)Tryptophan at and c) Thioflavin T at 25 0c and three different pH values. The fluorescence intensities for these three were measured at 303, 348, 482 nm respectively, in all the experiments.
There was slight increase in Tryptophan fluorescence at pH 5.0 and 7.0.
Consequently, a destabilized protein level in the body, through inappropriate nutrition or through effects of diseases adversely affects body processes. The study of protein concentration in the body, as well as in substances is therefore fundamental to nutritionists and health care professionals.
This report examined the features of E coli in respect to the expression systems with an emphasis on the limitation number that have been addressed. For a high gene dosage to be achieved, the heterologous cDNAs are normally cloned into replicate plasmids in a fashion that is relaxed and are normally existence at 15-60 copies per cell.
With respect to their structure, biochemists often refer to four different aspects:
Secondary structure, local interactions held together by hydrogen bonds between the lone pair of electrons of an oxygen atom and the hydrogen attached to a nitrogen atom.
4. If a particular protein was absent, it would lead to errors in protein synthesis. Errors in protein synthesis disrupt cellular fitness, cause disease phenotypes, leads to loss of function of the protein (non-functional proteins) and protein misfolding.
After the 30 minutes of incubation the rank is removed from the water bath and the samples allowed to cool.1 ml of each reaction is transfered to a 1 ml cuvette and A562 in a spectrophotometer read. The spectrophotometer zero is set to
Moreover, the GFP (Green Fluorescent Protein) gene usually exists naturally in the form of specialized photogenic cells found in the jellyfish Aquorea Victoria umbrella. This fluorescent protein can also be expressed in the bacteria, Escherichia coli. When the fluorescent protein gets exposed to U.V light source of long wave band, it produces a green light.
Proteomic is generally defined as the direct analysis of proteins in terms of their presence and relative abundance. Gel electrophoresis is a significant methodology employed for extraction of proteins in proteome analysis. The most commonly used technique in gel electrophoresis is Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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