Friday, February 27, 2009

Types of Protein based products based upon their derivation

Proteins, as biological macromolecules, are very important for the existence of normal activity in a living system. Because of the multifaceted role of proteins, they are also important commercially. Proteins may be classified, according to their derivation as described here:

1) Blood-derived Proteins and Vaccines:
Blood is the fluid connective tissue of humans. It is the most important medium for the transportation of gas and nutrients between tissues and sources. The blood forms the source for a number of therapeutically important proteins. A detailed understanding of the formation of blood cells (hematopoiesis) and detailed studies regarding the coagulation of blood have shown the presence of several useful proteins. In addition to blood-coagulating factors, whole blood and blood plasma have been used traditionally as blood products. These products are obtained from human volunteers who donate blood. But today some of the proteins of blood are produced by a recombinant technique. The blood coagulating factor known as factor VIII used in the treatment of hemophilia A and another factor, the factor IX for treating hemophilia B, etc., and the vaccine for hepatitis B are now synthesized by transgenic bacteria in fermentors.

2) Therapeutic Antibodies and Enzymes :
Antibodies and enzymes that are used for clinical applications are known as therapeutic antibodies. Polyclonal antibodies and monoclonal antibodies are examples. In tissue and organ transplantation, the body will recognize these as foreign objects and will produce antibodies against these tissues and organs leading to the failure of organ transplantation. Certain specific antibodies can be used against these natural antibodies and can revert the rejection process. For example, OKT-3 is used during kidney transplantation to revert the acute organ rejection in patients. Similarly, ReoPro is used to prevent blood clots. A large number of therapeutic antibodies are now produced by recombinant DNA techniques. Tissue plasminogen activator (t-PA) is an example of therapeutic enzyme used for acute myocardial infarction. There are a number of enzymes that are used as drugs. Asparaginase is used against some types of cancers and DNase is used for the treatment of cystic fibrosis.

3) Hormones and Growth Factors as Therapeutic Agents:
There are a number of proteins and peptides that act as hormones and growth factors. In the case of diseases due to metabolic errors such as diabeties, caused by the absence of these peptides, the concerned molecules can be administered to correct the metabolic errors. Insulin is the best example. In earlier times, insulin was prepared from the pancreas of cows and pigs. But now, human insulin is prepared through genetic engineering, by transferring the gene of insulin into bacteria; the transgenic bacteria will produce the active human protein. This insulin is called humulin. The methods of protein engineering have helped to develop altered forms of insulin, which are more active than the normal ones. There are a number of growth factors such as EGF (Epithelial Growth Factor) and plateletderived growth factors that are used for therapeutic purposes. EGF is used for the treatment of burns and injuries, during skin transplantation, for its growth. The platelet-derived growth factors are used for the treatment of diabetic and skin ulcers. A large number of growth factors and hormones are under clinical trials.

Tags: Bio Technology, Bio Genetics , Protein Products

Types of Protein based products based upon their Commercial Importance

Proteins being biological macromolecules can be classified, according to their commercial importance as described below :

1) Regulatory Factors :
Regulatory factors are also like growth factors, which are closely involved in the process of signal transduction and expression of specific genes. These factors are called cytokines because they are involved in the growth and proliferation of cells. Cytokines include interferones (INF), interleukins, tumor necrosis factors, colonystimulating factors, etc. All these factors find use in therapeutic application for the treatment of different diseases, both infectious and non-infectious. For example, (X-interferon is used in the treatment of hepatitis C, p-interferon is used for the treatment of multiple sclerosis, and y-interferon is used for severe granulomatous disease.

2) Proteins and Enzymes Used in Analytical Applications:
In addition to the use of antibodies and enzymes as therapeutic agents, they are also used in the diagnosis of diseases as the components of some confirmatory tests of certain diagnostic procedures. Hexokinase and glucose oxidase are used in the quantification of glucose in the serum and urine. Glucose-oxidase is used in glucose electrodes. Uricase is used for the estimation of uric acid present in urine. Alkaline phosphatase, horseradish peroxidase, and antibodies are used in ELISA (Enzyme Linked Immunosorbent Assay).

3) Industrial Enzymes and Proteins:
Among commercially useful proteins, industrial enzymes have the first place. Industrially useful enzymes include carbohydrate-hydrolyzing enzymes such as amylases, cellulase, invertases, etc., proteolytic enzymes such as papain, trypsin, chymotrypsin, etc., and other bacterial and fungal-derived proteolytic enzymes and lipases that can hydrolyze various types of lipids and fats. All these enzymes are important in the food and beverage industries, the textile industry, paper industry, and detergent industry. Proteases have a special use in the beverage industry, meat and leather industries, cheese production, detergent industry, bread and confectionary industry, etc. Various types of lipases are used for the modifications of various types of lipids and fats, production of various organic acids including fatty acids, in detergents, production of coco butter, etc. In addition to all these, enzymes are used in chemical industries as reagents in organic synthesis for carrying out stereospecific reactions.

4) Non-catalytic Functional Proteins:
These commercially important proteins are used in the food industry as emulsifiers, for inducing gelation, water binding, foaming, whipping, etc. These non-catalytic functional proteins are classified as whey proteins. The proteins that remain in solution after the removal of casein are by definition called whey proteins.Commercially-available whey protein concentrates contain 35 to 95% protein. If they are added to food on a solid's basis, there will be large differences in functionality due to the differences in protein content. Most food formulations call for a certain protein content and thus whey-protein concentrates are generally utilized as a constant protein base. In this case the differences due to protein content as such should be eliminated. As the protein content increases, the composition of other components in the whey-protein concentrate must also change and these changes in composition have an effect on functionality.

5) Nutraceutical Proteins:
Nutraceutical proteins represent a class of nutritionally-important proteins having therapeutic activity. The whey-protein concentrates and some of the milk proteins of infant foods contain certain pharmaceutical proteins having high nutritive quality. Infants get the required proteins from the mother's milk, which also contains certain therapeutic proteins that protect the baby from infection and other problems. There are other infant foods, which also have more or less the same composition as that of mother's milk, made up of cow's and buffalo's milk. All these food proteins provide the infants the raw building materials in the form of essential amino acids and at the same time protects them from microbial infections and other diseases. Thus, milk is a very good source of nutrition.

Tags: Bio Technology, Bio Genetics , Protein Products

Mass spectrometry for protein identification

There are various types of mass spectrometry depending on the type of ionization source or the ion analyzers. They are used for specialized studies in proteomics such as identification of proteins by generating mass fingerprints or the ionization pattern of the protein molecule, amino acid sequencing of peptides, and thereby the detailed studies of the three-dimensional structure of the protein and modification of the proteins if any can be detected very easily and efficiently. Some of the common types of mass spectrometry are discussed below.

MALDI-TOF Mass Spectrometry :
Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) is another method of ionization that does not require any heating for the production of ions and can be used for heat-sensitive molecules. In this method, the sample to be analyzed is mixed with a matrix compound and crystallized. The compound of this matrix is usually a weak inorganic acid. This mixture (matrix mixed with sample) is then excited with a laser, which results in the evaporation of the matrix compound. The matrix compound also carries the sample molecules into the vapor phase, resulting in indirect vaporization of the sample. Sample ions are formed by the exchange of electrons and protons with the matrix compound.

MALDI is especially useful for protein and peptide identification using masses alone, since the masses of ions can be determined with great accuracy. Time of flight is the technique used for the sorting and separation of ions generated by laser ionization.

Electrospray Ionization (ESI):
This is a newer method of ionization that does not cause excessive fragmentation. ESI generates ions directly from the solution without heating. Therefore, this method can be used for heat-sensitive molecules, which cannot be ionized by heating. The sample is finely sprayed in the presence of an electrical field. Charge accumulates on the sample droplets, which then explode due to mutual repulsion of charges leading to the formation of ions. Both single and multiple-charged ions can be produced in this manner.

Tandem Mass Spectrometry (MS/MS or Two-dimensional MS):
Tandem MS is a procedure in which multiple rounds of mass spectrometry are performed to obtain greater resolution of the ions generated by the unknown sample molecule. Short stretches of polypeptides can be sequenced with tandem MS. In general a polypeptide is cleaned into a number of fragments enzymatically and the products are taken into the mass spectrometer for analysis one by one to generate the primary structure or the amino acid sequence.

The protein solution is cleaned into short stretches of pep tides preferably by an enzyme such as trypsin or chymotrypsin. The mixture of short pep tides thus generated is injected into the tandem MS for analysis. The tandem MS is an instrument with two MS in a series. They are designated as MS1 and MS2. In the first mass analyser (MS1) the peptide mixture (precursor ions) is sorted resulting in the selection of only one peptide from the several types of pep tides produced by cleavage, which comes out at the outlet of the MSl. The selected peptide (precursor ion) is then transmitted to a collision cell where it undergoes collision-induced dissociation (CIO). The peptide is further fragmented by a high-energy impact with a small amount of inert gas such as helium or argon known as the collision gas. The fragment ions, thus produced, are transmitted to the second mass analyzer (MS2), which measures the m/z ratio of all the charged fragments. During the process of fragmentation the peptide molecule breaks at the peptide bonds resulting in the product ions. This fragmentation does not involve the addition of water as in the case of enzymatic cleavage.

The MS2 scans the product ions and generates one or more sets of peaks. A set of peaks consists of all the charged fragments (product ions) generated by breaking the same type of bond (peptide bond) by CIO. Each peak in a set represents one amino acid less than the previous peak. The difference in mass (m/z) between two adjacent peaks indicates the amino acid that was lost during CID, thus revealing the sequence of amino acids of the peptide. This type of mass spectrometry, also known as 2D MS, is usually used for the analysis of complicated mass spectrum, particularly useful in the amino acid sequencing of pep tides and post-translational modifications.

Tags: Bio Technology, Bio Genetics , Protein Purification

Process of Characterization of Proteins

The purified protein needs to be identified and characterized. There are various methods to accomplish this. The purity of the protein can be determined by the SDS-PAGE or by isoelectric focusing (IEF) or by two-dimensional gel electrophoresis.

Identification or characterization of protein can be carried out with the help of anyone of the following techniques or by a combination of these:

1) Two-dimensional gel electrophoresis,
2) Peptide fingerprinting,
3) Protein sequencing, and
4) Mass fingerprinting by mass spectrometry. Here we are discussing the technique of Mass Spectrometry.

Mass Spectrometry: It is a chemical-analysis technique that is used to measure the mass of unknown molecules by ionizing, separating, and detecting ions according to their mass-to-charge ratios. Mass spectrometry is also used to determine the structure of molecules.Mass spectrometry helps us in the following areas:

1) To determine the sequence of proteins and peptides.
2) To identify the structure of other biomolecules such as lipids, carbohydrates, oligonucleotides, etc.
3) To detect the presence of banned substances in athletes.
4) To determine the composition of rocks and thus determine their age and origin.
5) To identify isotopes of various elements.
6) To perform forensic analysis to detect the presence of certain substances.

A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its chemical structure. There are different types of mass spectrometers and sample introduction techniques, which allow a wide range of analyses. This discussion will focus on the principle of mass spectrometry and its use in the study of protein chemistry. The techniques can be used to sequence peptides and proteins and to study their interactions. It is useful in the identification of protein molecules, their accurate molecular weight, the post-translational modifications, and structural functional relationship of proteins.

It is now possible through mass spectrometer to 'see' proteins of molecular weight as high as 100,000 daltons. A big advantage is that a very small sample, as small as picomoles, is required. Thus, it is possible to do two-dimensional gel electrophoresis of proteins of a cell (to separate the few thousand different proteins) and identify them using mass spectrometer. This approach of protein analysis and identification is one of the important techniques of what is called proteomics-the study of the complete protein complement of a cell.

Let us understand as to what is a Mass Spectrum:
A mass spectrum is a plot that shows the relative abundance of ions of various mass-to-charge ratios. The X-axis represents the mass-to-charge ratio of the ions and the Y-axis represents the relative abundance of each ion.

Mass spectrometry can be in association with or linked to one or more separating techniques. For example, in organic chemistry mass spectrometry is always linked to Gas chromatography and therefore, is called GC-MS. Gas chromatography will convert the samples into gaseous phase, which is introduced to the mass spectrometer. In addition to this, GC can be used to separate the components in a mixture and the separated pure compounds can be directly identified and analyzed by a mass spectrometry spectrum. This is the advantage of linking a separating technique to mass spectrometry. Similarly, in the case of protein studies mass spectrometry can be linked to various types of liquid chromatographic systems such as GPC, ion exchange, or affinity chromatography individually or in tandem, or to capillary electrophoretic systems, which can also be operated automatically, like HPLC.

Tags: Bio Technology, Bio Genetics , Protein Characterization

Monday, February 23, 2009

How do we scale up the Protein Purification Process

When the protein purification protocol forms the part of an industrial process for the production of industrially important or pharmaceutically important protein such as vaccines, hormones, etc., the laboratory scale protocol also has to be scaled up. Actually, it forms the process of downstream processing.

Special care should be taken to bring the cost of production of the protein down by purchasing the chemicals in bulk quantities and using equipment and holding vessels, which are permanent or recyclable. The materials and vessels should be made up of cheap materials and should be inert and resistant to corrosion. Leaching of toxic metals and chemicals into the product should be avoided. The process of manufacturing and downstream processing require the approval of the regulatory authority to produce and market a protein that is useful in food industry or as pharmaceuticals. About 80% of production costs is for downstream processing and quality assurance. Therefore, by controlling the various steps and equipment in downstream processing and product recovery, we can bring down the cost of production without compromising the quality of the product.

Bulk-Protein Production :
The protein-purification procedure that is applicable for the laboratory scale is applicable for the downstream process of the industrial scale also. The process can be used for the purification of enzymes in bulk quantities not only from microbial fermentation systems but also from plant and animal tissues.

Proteins for Therapeutic and Diagnostic Purposes:
Drug proteins such as digestive enzymes, therapeutic proteins such as antibodies or vaccines, hormones, growth factors, etc., and other diagnostic proteins should be purified to a very high degree. Those proteins that are for pericutaneous or intra-venal administration should be highly purified and sterile. Parentral preparations are those that are intended for intra-venal administration or for infusion or for implantation. Such materials should be highly purified and sterile.

Tags: Bio Technology, Bio Genetics, Protein Purification

Monday, February 16, 2009

Extraction & Purification of Proteins

Biochemists investigate proteins at different levels. At the simplest level they carry out qualitative chemical tests to find out if samples of material contain protein of any sort. At the other extreme they can use the most up-to-date technology to find out the precise arrangement of every atom in each molecule of a particular protein. For structural elucidation, proteins should be very pure homogeneously and in some cases they should be crystallized. There are various methods of extraction and purification of proteins from different sources such as microbes and plant and animal tissues. Extraction and separation techniques for the purification of compounds from microbial cultures and plants and animal tissues are collectively called downstream processing. For industrial purposes, there are some microorganisms identified as non-pathogenic, non-toxic which do not produce any antibiotic. These microorganisms can be used as a source of industrial enzymes and proteins as well as for introducing foreign genes for producing recombinant proteins. Such microorganisms are called generally regarded as safe (GRAS) microbes.

Plants and some animal tissues such as pancrease form the source of some important industrially important proteins and enzymes, which are used in food industry and medicine. Such enzymes should be extracted from non-toxic plant parts and the animal tissues used should be free from infectious diseases. For example, one of the important industrial enzymes, papain, is extracted from the latex of green leaves and fruits of papaya. Papain is used in the meat industry and leather industry for meat tenderization, processing the collagen and other fibrous proteins present in leather, clarification of beverages, and also in medicines as a digestive aid and for cleaning wounds.

Proteins and enzymes of animal origin can be extracted and purified from the respective organs in which the enzyme is present in higher quantities. Slaughterhouses are one of the centers for the supply of tissues and organs necessary for the extraction of certain proteins such as insulin. Traditionally, insulin was obtained from the pancreas of cows and pigs. It requires the slaughtering of about 100 to 150 pigs or 15 to 20 cows to meet the insulin requirement of a single diabetic patient per year. From this we can imagine the number of animals that have to be killed to meet insulin requirements. But modern biotechnology and genetic engineering has come in to help the situation.

Nowadays we don't depend on animals for insulin. There are a number of pharmaceutical companies which manufacture and market human insulin produced by genetic engineering. Similarly, a large number of therapeutic proteins such as vaccines and hormones are now produced by genetic engineering. Today, efforts are there to produce transgenic plants and animals capable of producing these therapeutic antibodies and hormones, and other industrially important proteins in specific organs of plants and animals. For example, the production of edible vaccines (edible plant parts such as fruits and tubers containing the vaccine) and expression of certain proteins such as insulin in the milk of cows and goats, and in eggs. This method of genetic engineering for large-scale production of specialized proteins and other molecules is called molecular farming. The main advantages of molecular farming is that the cost of production can be reduced, costly and time-consuming fermentation procedures and downstream processes can be avoided, large-scale production of the specific compounds is possible, and ease of production and purification procedures is advantageous.

Edible vaccines have the advantage that they can be stored for a long time without refrigeration, can be easily transported, and can be administered by feeding the fruit or the plant parts having the vaccine. While eating the vaccinated fruit, the vaccine molecules will be absorbed into the bloodstream through the mucous membrane lining the mouth and esophagus. Even animals can be vaccinated by this way. Attempts are being made to develop transgenic fodder grass containing the anthrax vaccine. Cattle can be fed with this transgenic fodder grass and be vaccinated effectively against anthrax.

Tags: Bio Technology, Bio Genetics, Proteins

Understanding Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a method for the separation and identification of proteins in a sample by displacement in two-dimensions oriented at right angles to one another. This allows the sample to separate over a larger area, increasing the resolution of each component.

Two-dimensional gel electrophoresis is generally used as a component of proteomics and is the step used for the isolation of proteins for further characterization by mass spectroscopy. In the lab we use this technique for two main purposes. Firstly, for the large-scale identification of all proteins in a sample. This is undertaken when the global protein expression of an organism or a tissue is being investigated and is best carried out on model organisms whose genomes have been fully sequenced. In this way the individual proteins can be more readily identified from mass spectrometry data. The second use of this technique is differential expression; this is when two or more samples are compared to find differences in their protein expression.

Two different protein-separating techniques are combined in sequence to achieve the goal of protein separation and identification-Iso electric Focusing (IEF) and SDS-PAGE. Isoelectric focusing (IEF) is used in the first-dimension. This separates proteins by their charge (pI) and SDS-PAGE (sodium dodecyle sulphate-polyacrylamide gel electrophoresis) is used in the second-dimension. This separates proteins by their size (molecular weight, MW). The procedure is known as ISO-OALT (iso for isoelectric focusing and dalt for molecular weight in dalton).

Isoelectric focusing (IEF). The side chains of amino acid residues of a protein contribute a net charge for protein molecules, which depend on the pH of the medium. In simple electrophoresis the mobility of the protein molecules is dependent on its charge that is controlled by the pH. There is a pH for every protein molecule at which the net charge of the protein becomes zero. This pH is known as isoelectric pH or isoelectric point (PI). At isoelectric pH the protein loses its mobility in the electric field. The technique of protein separation based on the property of isoelectric pH or PI is known as isoelectric focusing (IEF). A pH gradient is generated on an IEF gel and proteins are allowed to move in an electric field and that results in the separation of an individual protein species according to its isoelectric point.

Tags: Bio Technology, Bio Genetics, Proteins

Sickling of Cells & Malaria

The high representation of the hemoglobin S gene in some populations reflect the protection it provides against malaria. The malaria parasite does not survive as well in the erythrocytes of people with the sickle trait as it does in the cells of normal people. The basis of the toxicity of sickle hemoglobin for the parasite is unknown. One possibility is that the malarial parasite produces extreme hypoxia in the red cells of people with the sickle trait. These cells then sickle and are cleared (along with the parasites they harbor) by the reticuloendothelial system. Another possible mechanism is that low levels of hemichromes are formed in sickle trait erythrocytes. Hemichromes are complexes containing heme moieties that have dissociated from the hemoglobin. Hemichromes catalyze the formation of reactive oxygen species, such as the hydroxyl radical, which can injure or even kill malarial parasites.

The malaria hypothesis maintains that during prehistory, on average, people without the sickle gene died of malaria at a high frequency. On the other hand, people with two genes for sickle hemoglobin died of sickle cell disease. In contrast, the heterozygotes (sickle trait) were more resistant to malaria than normal people and yet suffered none of the ill effects of sickle cell disease. This selection for heterozygotes is called balanced polymorphism. Support for this concept comes from epidemiological studies in malaria-endemic regions of Africa. The frequency of the sickle cell trait is lower in people coming for treatment to malaria clinics than is seen in the general population. The reasonable assumption is that relative protection from malaria is at work in this situation.

Although malaria remains a major health problem in many tropical regions of the world, the disease is not a significant threat to people in temperate zones. Consequently, the protection afforded by the sickle trait no longer has a survival advantage for many groups of people in whom the sickle cell gene is common. This has left sickle cell disease the major health issue in these populations.

Tags: Bio Technology, Bio Genetics, Siclkling of cells

Understanding of Protein Fingerprinting Technique

Breaking the protein molecules down into shorter fragments called peptides, Pauling subjected these fragments to another technique called protein fingerprinting by paper chromatography.

The technique of protein fingerprinting involves the following steps:

1) Extract and purify hemoglobin from sickle cell RBC and normal RBC separately in a clean test tube.

2) Digest these proteins with a commercial sample of trypsin separately under standard conditions. Trypsin is another type of serine protease that cleaves the peptide bond adjacent to a lysine or arginine residue in a protein molecule.

3) The cleaved peptides are subjected to paper electrophoresis under pH (pH 2.5) and dry the paper.

4) After electrophoresis they are subjected to paper chromatography perpendicular to the direction of electrophoresis using the solvent system water: butanol: acetic acid in the ratio 5:4:1. The peptides will separate depending on their partition coefficient, which further depends on their degree of hydrophobicity. The more hydrophobic peptide will move fast and the less hydrophobic will move slowly.

5) Remove the chromatographic paper and stain with ninhydrin.

6) Examine the peptide spots and compare with the standards.

When this procedure is applied to samples of normal and mutant (sickle) hemoglobin molecules (alpha and beta chains) that had been broken down into specific pep tides, all the spots are the same except for one crucial spot, which represents the difference between sickle cell and normal hemoglobin.

The protein fingerprinting or the peptide mapping developed for the molecular studies of sickle cell hemoglobin became a very powerful technique for the identification of protein samples from different sources. The peptide fingerprint of a protein from new sources can be compared with that of the standard protein and thus,. the variations can be identified or understood. This simple technique of peptide fingerprinting has given rise to another similar and more powerful technique: two-dimensional gel electrophoresis. This is a combination of two electrophoretic techniques - Isoelectric focusing and SDS-PAGE-in a series. First, the protein is subjected to isoelectric focusing, which is followed by 50S-PAGE in a direction perpendicular to the first. This technique was found to be very useful for proteomes studies, expression of protein profiles of cells grown under different conditions (for example, normal cells and diseased cells), and also easy identification of proteins in combination with mass spectrometry. All these technological advancements including amino acid sequencing have provided an enormous quantity of data, and that has given rise to computerized databases and homology searches and protein identification. All these have led to a generation of bioinformatics and computational biology.

Tags: Bio Technology, Bio Genetics, Proteins

Sickle Cell Anemia the cause of oxygen depletion

Sickle cell anemia, creates serious depletion of oxygen through two mechanisms:
1) Because of molecular changes within the sickled cell, oxygen-carrying capacity of the blood is greatly reduced.

2) Because of their peculiar shape, greater rigidity, and tendency to stick together, sickle cells clog smaller vessels in the circulatory system-the arterioles and capillaries, in particular-preventing the blood from delivering oxygen and nutrients, and removing carbon dioxide and wastes from tissues.

This disease was reported for the first time by a Chicago-based cardiologist James B. Herrick in 1910. It was recognized to be the result of a genetic mutation, inherited according to the Mendelian principle of incomplete dominance. Initially, it was not clear what the actual defect was, that caused the sickling. Various experiments indirectly narrowed down the site of the defect to the hemoglobin molecule. The most direct evidence that mutation affected the hemoglobin molecule came from electrophoretic analysis, a method to separate complex mixtures of large molecules by means of an electric current on a gel. When hemoglobin from people with severe sickle cell anemia, the sickle cell trait, and normal red blood cells was subjected to electrophoresis.

It was clear that hemoglobin molecules of people with sickle cell anemia migrated at a different rate, and thus ended up at a different place on the gel, from the hemoglobin of normal people. What was even more interesting was the observation that individuals with the sickle cell trait had about half normal and half sickle-cell hemoglobin, each type making up 50% of the contents of any red blood cell.

To confirm this latter conclusion, the electrophoretic profile of people with the sickle cell trait could be duplicated simply by mixing sickle cell and normal hemoglobin together and running them independently on an electrophoretic gel. These results fit perfectly with an interpretation of the disease as inherited in a simple Mendelian fashion showing incomplete dominance. Here, then, was the first verified case of a genetic disease that could be localized to a defect in the structure of a specific protein molecule. Sickle cell anemia thus became the first in a long line of what have come to be called molecular diseases. Thousands of such diseases (most of them quite rare), including over 150 mutants of hemoglobin alone, are now known.

Tags: Bio Technology, Bio Genetics, Proteins

Importance of Structure on Function of Proteins

Proteins are the workhorses of cells and in every activity there is the involvement of one or more proteins in different ways. Now, it is very clear that each protein has a specific three-dimensional shape determined by the amino acid sequence and various other intermolecular interactions. This three-dimensional shape has a great influence on the biological function that it performs in the cells. We consider two proteins, as an example, to understand the importance of a three-dimensional structure on its specific function: a proteolytic enzyme, chymotrypsin, and the oxygen carrying protein, hemoglobin.

Chymotrypsin - A Protein- Digesting Enzyme:
Chymotrypsin is a member of a family of enzymes, all of which cleave peptide bonds through the action of an active site serine (the serine proteases). This family includes the pancreatic enzymes chymotrypsin, trypsin, and elastase as well as a variety of other proteases (e.g., cocoonase, thrombin, acrosomal protease, etc.). Chymotrypsin, trypsin, and elastase show a high degree of similarity in their overall tertiary structure, but have different substrate specificities determined by a specific substrate-binding site on each enzyme.

Chymotrypsin is one of the proteinhydrolyzing enzymes produced by the digestive gland pancreas. The protein present in the food that we eat is digested mainly by two proteases - trypsin and chymotrypsin in the beginning of the small intestine (the duodenum). These two digestive enzymes are produced by pancrease and are released into the duodenum through the pancreatic duct. Thus, the site of production of these enzymes is the pancreas and their site action is the duodenum.

Trypsin and chymotrypsin cut the linear polypeptide chains into short peptides by cutting at specific sites. These short pep tides thus produced are acted upon by other peptidases releasing amino acids. But the pancreas is made up of many proteins. How are these proteins protected from the hydrolytic activity of chymotrypsin? These types of hydrolytic enzymes, particularly proteases are produced in an inactive form called zymogen and are transported to the site of action, the duodenum-where it is converted into an active enzyme by a process known as in situ activation. Because of this process, the protein undergoes a major change in its three-dimensional shape, which is now suitable for its interaction with its substrates. The active chymotrypsin enzyme is known as alpha. chymotrypsin and its inactive form from which it is produced is called chymotrypsinogen.

Know the Chymotrypsinogen:
Chymotrypsinogen, the precursor (zymogen) of active chymotrypsin, consists of 245 amino acid residues. Activation of chymotrypsinogen involves proteolytic cleavage at two sites along the chain and removal of two amino acids at each cleavage site. The resultant three peptide chains are A, B, and C. These three chains are held together by five disulfide bonds and fold into a globular structure. This process of folding brings three distantly placed amino acid residues his 57, asp 102, and ser 195 close together in a particular order to form the active center or the reaction center of the enzyme. The overall chymotrypsin molecule is folded into two domains, each containing six beta strands arranged as antiparallel sheets that form a circular structure known as a beta barrel. The active site residues (ser 195, his 57, and asp 102) are far apart in the primary sequence but are brought together in a crevice formed between the two protein domains

Sunday, February 8, 2009

Levels of Complexities of Structure of Proteins

Proteins and peptides are biopolymers composed of amino acid residues interlinked by amide bonds. Their structures can be discussed in terms of four levels of complexity and are as follows:
1) Primary structure
2) Secondary structure
3) Tertiary structure
4) Quaternary structure

The linear unbranched chain of amino acids linked together by covalent bonds known as peptide bonds is called the primary structure of a protein. In addition to peptide bonds, other types of covalent linkages such as disulphide bonds, if present, are also included in the primary structures. The types and the sequence of amino acids present in the polypeptide chain determine the nature of the secondary structures at different regions of the chain. The secondary structure of a segment of a polypeptide chain is the local spatial arrangement of its main-chain atoms without regard to the conformation of its side chains or to its relationship with other segments.

The major types of secondary structures observed in protein molecules are alpha (a ) helices and beta (b ) pleated sheets in addition to random coils and beta turns. All these secondary structures may be present independently or may be together in the secondary or tertiary structures of a single polypeptide chain. The secondary structure undergoes further folding and reorganization within the molecule resulting in higher order compact structures or the tertiary structure.

There are structural components comprising a few alpha-helices or beta-strands, which are frequently repeated within structures, called supersecondary structures (being intermediate to secondary and tertiary structures). These compact structurally distinct elements are known as motifs. When these structurally distinct regions of protein molecules are associated with a specific function, those structurally and functionally distinct units are called a domain. Structurally-related domains are found in different proteins, which perform similar functions.

The molecular forces, which are responsible for the secondary and tertiary structures, are the non-covalent interactions between the various amino acid side chains within the molecule and with the water molecule surrounding it. The main molecular force responsible for the various secondary structures are the hydrogen bonds and the molecular forces behind the tertiary structures are the ionic bonds, hydrogen bonds, hydrophobic and hydrophilic interactions, and van der Waals force. Secondary and tertiary structures represent the most thermodynamically stable conformations or shapes for the molecule in a solution. The quaternary structure is the assembly of two or more independent polypeptides or proteins at their tertiary stage to form a multimeric protein. The individual component pep tides of the multimeric proteins are known as subunits and are held together via non-covalent forces. The subunits of a multimeric protein may be similar or dissimilar. For example, hemoglobin contains four polypeptide chains (20. chains and 213 chains) held together non-covalently in a specific conformation as required for its function.

The major molecular forces that cause the linear polypeptide chain to undergo a specific type of coiling and folding in space to a characteristic three-dimensional shape are the non-covalent forces. These forces, to a greater extent, lie in the chemical and structural properties of the constituent amino acid residues of the polypeptide chain. There are 20 types of amino acids by which the entire protein of the living system is composed. These amino acids can be broadly classified into three categories-hydrophobic (tryptophan, phenylalanine, leucine, etc.), polar (glutamine, serine, etc.), and charged (aspartic acid, lysine, etc.) amino acids. Therefore, these amino acids are capable of interacting with each other within the protein molecules via various non-covalent interactions leading to a very characteristic shape and biological property for the protein molecule.

Tags: Bio Technology, Bio Genetics, Proteins

An Introduction to Structure of Proteins

A single cell develops into a multicellular embryo through a large number of complicated biochemical reactions mediated and controlled by various types of proteins expressed during the course of its development. Every function in the living cell depends on proteins. They make us who we are and make our cells operate properly. A cell cannot function without proteins. The shape of a protein determines its biological activity. A single protein may have a varying structure and more than one function. Proteins have many different biological functions. Proteins are even classified according to their biological roles. The key to appreciating how different proteins function in these different ways lies in an understanding of protein structure and their three-dimensional shape. Proteins interact with other molecules such as small molecules, other proteins, nucleic acids, lipids, etc., and these interactions form the basis of their biological roles. Structural complementarity is the means of molecular recognition that allows molecules to interact. The structure of one molecule is complementary to that of its partner(s) in the interaction, like pieces in a puzzle, or a lock and its key. Proteins, by virtue of their architectural diversity, are ideal for such complementary interactions. In short, the structure or the molecular shape of the protein determines its function. Therefore, to understand the function and biological role of a protein it is essential to understand the structure and three-dimensional shape of the protein in detail.

The detailed study of the structure of proteins requires protein extraction and purification to its homogeneity. The purified protein has to be analyzed by various biochemical and instrumental methods to get the details about its chemical composition. The pure protein obtained has to be crystallized to study its three-dimensional shape by x-ray crystallography (an x-ray diffraction technique). Another equally powerful technique to elucidate the three-dimensional structure of protein is NMR spectroscopy. The structural study of protein, thus has two parts-the first part is determination of amino acid sequence and the second part is the elucidation of the three-dimensional shape of the protein formed by the specific folding of the polypeptide chain controlled by a number of molecular forces. The extraction and purification and its crystallization are the preconditions for the detailed structural and functional studies.

The ability to sequence polypeptides was a major step forward in the understanding of the relationship between protein structure and function. It was Dr. Frederick Sanger who developed the basic chemical method for sequencing proteins during the 1940s. He showed for the first time, that proteins are a linear polymer of amino acids, linked in a continuous sequence by peptide bonds. He received the Nobel Prize in 1958 for determining the sequence of the peptide hormone insulin. The peptide bond is formed between the alpha-amino and alpha-carboxyl groups of two adjacent amino acids. Pehr Edman modified this process of amino acid sequencing by introducing a new reagent-phenyl-isothiocyanate for the sequential removal of amino acids and their identification in a protein. This method of sequencing is now automated and called the Edman Degradation Reaction and the instrument is called the Sequenator.

Tags: Bio Technology, Bio Genetics, Proteins

Expression of Proteins in Cells

The proteins, which are produced, have various life spans or half-life ranging from a few seconds to many months or years.

Each protein can be identified by its unique amino acid sequence, the three-dimensional shape of the protein (that mainly depends on amino acid sequence and other environmental factors), function of the protein, and cellular location.

The proteins expressed in a cell may be intracellular (present within the cell) or extracellular (secreted outside the cells), or circulating in the blood like hormones, immunoglobulins, etc., constantly interacting with other molecules such as proteins, lipids, sugars, DNA, RNA, metal ions, vitamins, etc., or with other cells. The functions of these circulating proteins are also influenced by their interaction with other molecules that are present nearby. Even though the total number of genes estimated is about 35,000, the actual number of proteins is much higher than this. About 17, 000 proteins were identified at the gene level but information regarding their function and biological role is still being investigated in detail.

The type and total number of genes in an organism will be stable (static) and are identical in all somatic cells of an organism. But, the total number of proteins expressed by a cell (protein profile) of an organism is always variable (dynamic). The protein profile of a cell depends on its metabolic state, stage of development, and other micro- and macro-environmental factors, which influence the expression of a set of genes at a particular time. Therefore, the challenge for the future is to determine the actual total number of proteins expressed in each cell type and find out the functions and biological role of these proteins in metabolism, health, and disease. These topics provide an exposure to the three-dimensional structure of proteins and their relationship to proteomics, recombinant DNA technology, genomics, and finally bioinformatics.

Tags: Bio Technology, Bio Genetics, Proteins

Understanding the Broad Compositions of Proteins

In spite of these diverse biological functions, proteins have relatively homogeneous compositions. All proteins are linear polymers of the same 20 types of amino acids in different combinations. The major difference between proteins is in the sequence in which the amino acids are assembled into polymeric chains. The secret to their functional diversity lies partly in the chemical diversity of the 20 amino acids, but primarily in the diversity of the three-dimensional structures that these amino acid building blocks can form by linking in different sequences. The amazing functional properties of proteins can be understood only in terms of their relationship to the three-dimensional structures of proteins.

Now we know that the amino acid sequence of a protein and thereby its three-dimensional structure is specified by a gene. But, this is not completely true. Even though the gene sequence specifies the amino acid sequence of the protein, the three-dimensional structure is also influenced by a number of other factors. The number of proteins produced in a system always exceeds the number of genes. The Human Genome Project has announced the presence of about 35,000 genes. But the actual number of proteins encoded by these genes exceeds the number of genes.

This is mainly because of the various types of molecular modifications such as deletion of amino acids, chemical modifications of certain amino acids, addition of other macromolecules and groups such as phosphate groups (phosphorylation), acetyl groups (acetylations), sugar and other types of carbohydrates (glycosylations), lipids, etc. All these chemical modifications of proteins just after their formation are collectively called post-translational modifications. Actually, these post-translational modifications are responsible for the diversity in the three-dimensional structures and functions along with the amino acid sequence prescribed by the respective genes. A number of proteins are expressed in all cells irrespective of their functional specialization. Such proteins are called housekeeping proteins, required for the basic life activities of all cells_ But there are certain proteins, which are unique to certain cells. Hemoglobin in erythrocytes (RBC), collagen, myosin, etc., in muscle cells are some examples. This is called cell-specific or organ-specific or tissue-specific gene expression. The expressions of these genes are under the control of very specific regulatory proteins or other types of small molecules called transcription factors.

Tags: Bio Technology, Bio Genetics, Proteins

Effect of Malfunctioning of Proteins in the Body

The absence or malfunctioning of one or more proteins in the system can cause serious life-threatening diseases. The malfunctioning of proteins can be traced to some type of structural abnormality due to variations in the chemical composition. For example, the absence of one of the subunit, beta chain of the oxygen-carrier protein hemoglobin of RBC, can cause thalassaemia.

This metabolic error due to abnormal hemoglobin affects many children who can only survive on repeated blood transfusion. Another type of abnormal hemoglobin is where the beta chain is mutated and the glutamic acid at position six is replaced with valine and results in deformed RBC and a condition known as sickle cell anemia.

The absence of an enzyme adenosine-deaminase, an important enzyme in nucleotide metabolism, can cause the disease known as SCID (severe combined immunodeficiency) in children. These children cannot survive infancy. There are some types of infectious protein particles known as 'prions', which can turn normal proteins to rogue proteins or incorrectly shaped proteins and can cause diseases such as mad cow disease. To understand more about the relationship between the disease and the structural abnormality of the protein we should know more about the structure and its relationship with biological activity.

Tags: Bio Technology, Bio Genetics, Proteins

Importance of Proteins for Life forms

Proteins are essential to maintain the structure and function of all life forms. The word 'protein' itself is derived from the Greek word protos, meaning "primary" or "first." Proteins are vital for the growth, repair, and maintenance of muscles, blood, internal organs, skin, hair, and nails, and their functions are endless. Each and every property that characterizes a living organism is affected by proteins, whether it is a bacteria or a human body. Nucleic acids, another major biological macromolecule, are also essential for life; they encode genetic information-mostly specific for the structure of proteins-and the expression of that information depends almost entirely on proteins. The fertilization of an egg with a sperm and the development and differentiation of the resultant zygote into a fully developed organism and its growth and maintenance of life activities up to its death is controlled and programed by a large number of proteins.

In our body, when we breath, oxygen present in lungs will be taken by the hemoglobin present in the RBC of blood to the various cells of the system for the process of cellular respiration. Movements and activities of body parts and systems including lungs, heart, stomach, etc. are happening due to the contractions and relaxations of various types of muscles. Myosin, actin, and collagen are the protein molecules involved in body structure, protection, and muscular contraction and relaxation. The structure of cells, and the extracellular matrix in which they are embedded, is largely made of protein. Plants and many microbes depend on carbohydrates such as cellulose for support. All biological activities of cells are mediated and regulated by a large number of catalytic proteins called enzymes. The function of the human brain and the speed at which the electric impulses are generated and transmitted to coordinate various activities of the systems are meticulously done by a large number of proteins that act as enzymes and receptors.

The receptors and hormones are another class of proteins, which act as signal molecules that are involved in the coordination of different metabolic functions of the system. There are the proteins called transcription factors, which turn the genes on and oft to guide the differentiation of the cells and development, and there are many more activities in which proteins are involved. Thus, proteins are diverse in their functions and are truly the physical basis of life. To understand the diversity in the biological function of proteins, their molecular structure and shape has to be studied in detail, since the function is closely related to the structure.

Tags: Bio Technology, Bio Genetics, Proteins

Tuesday, February 3, 2009

Understanding the methods of DNA Isolation

DNA isolation and purification is a technique used in laboratories engaged in molecular biology experiments. There are a number of standardized techniques and variations, which can be adopted according to the type of cells or tissues. The isolation and purification methods used in earlier times were lengthy and tiresome with the use of ultra-centrifugation. But now with the advancement of separation techniques, the procedure is very simple and short.

In any method of extraction and purification, there are three main steps:

1. Breaking of the cells
2. Extraction of DNA
3. Purification

Cells can be broken in different ways. One common method for lysis of bacterial cultures is alkaline lysis. In the case of animals, cells can be lysed by simple detergents or by hypotonic solutions. Plant tissues can be homogenized by strong detergents such as SDS and heating at high temperatures. But there are various types of DNA isolation kits marketed by a number of biotechnology companies, which are very simple, short, and easy to handle.

Isolation of Plasmid DNA by Alkaline Lysis Method:
This method is used for the large-scale isolation of plasmid and cosmid DNA by a modification of alkaline lysis procedure, followed by purification by phenol chloroform extraction. Cells containing the desired plasmid or cosmid are harvested by centrifugation, incubated in a lysozyme buffer (re-suspension buffer), and treated with alkaline detergent. The alkali breaks the cells and the DNA and proteins- are released into the medium. Detergent solubilizes the proteins and DNA. The proteins and membranes are precipitated with sodium acetate. The precipitate is centrifuged out at a higher RPM and the supernatant contains the DNA. Finally, the DNA is precipitated out by adding 95%. ethyl alcohol or propanol. The DNA pellet is resuspended in a Tris EDTA buffer. This DNA sample contains some DNA-binding proteins, which have to be removed. This can be carried out by phenol-chloroform extraction. There are several variations to this protocol, which is suited to the situations- and type of bacterial cultures.

Genomic DNA Isolation from Blood:
Genomic DNA isolation is performed according to the standard protocol suggested by Federal Bureau of Investigation, USA. After the blood samples (stored at -70°C in EDTA vacutainer tubes) are thawed, a standard citrate buffer is added, mixed, and the tubes are centrifuged. The top portion of the supernatant is discarded and additional buffer is added, mixed, and again the tube is centrifuged. After the supernatant is discarded, the pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is incubated at 55°C for one hour. The sample then is phenol-extracted once with a phenol/ chloroform/ isoamyl alcohol solution, and after centrifugation the aqueous layer is removed to a fresh microcentrifuge tube. The DNA is ethanol-precipitated, resuspended in buffer, and then ethanol-precipitated a second time. After the pellet is dried, buffer is added and the DNA is resuspended by incubation at 55°C overnight, and the genomic DNA solution is assayed by the polymerase chain reaction.

DNA Isolation from Plant Tissues:
Plant tissues bring up several problems during DNA isolation. Plant cells have a rigid cell wall and the tissue contains a number of toxic metabolites, which can interact with the DNA and change its nature and make it useless for other experimental purposes. Metabolites such as mucilage and other carbohydrates can very easily- form complexes with DNA and it can be damaged. Therefore, the extraction buffer should be supplemented with some compounds that can protect the DNA against these metabolites.

Many DNA-isolation techniques widely employed by plant molecular biologists use a CTAB (Cetyltrimethylammonium bromide) extraction buffer. This compound forms a complex with DNA and thus protects it from other toxic metabolites such as mucilage and phenolics.

The DNA, isolated and purified by any of these methods, can be used for a variety of experimental purposes. It can be used for restriction digestion analysis, cloning, ligation, transformation experiments, in vitro transcription, PCR amplification, RFLP (restriction length polymorphism), fingerprinting, RAPID (random amplification polymorphic DNA), sequencing, nick translation and radiolabeling, preparation of genomic DNA library and cDNA library, etc.

Tags: Bio Technology, Bio Genetics, DNA Isolation

Know the Pedigree Analysis of Humans

Humans are unique among organisms in many ways. But one way, which is near and dear to a geneticist's heart, is that humans are not susceptible to genetic experimentation. In practice, we humans actually share this characteristic with many long-lived organisms who delay first births. In short, it is not terribly convenient to perform experimental crosses if one has to wait 15 years between generations. However, for humans, one also has to add that our system of morality uniquely does not allow such experimentation on humans. This is an unfortunate state of affairs since there is no other organism for which practical knowledge of their genetics would be more useful, especially in the case of the genetics of heritable diseases. It has been found that human genetics may readily be inferred so long as good records have been kept within large families. This formal mechanism of inference is called pedigree analysis.

Here we are presenting many aspects of human genetics, with particular consideration to strategies of pedigree analysis whereby we are attempting to infer the genetics of human conditions based on knowledge of marriage (mating) and affliction in large extended families.

Procedures for Pedigree Analysis:
Pedigree analysis is one of the central tasks of the human geneticist. It involves the construction of family trees. Family history information is often collected at major family gatherings. A pedigree is used to trace inheritance of a trait over several generations.

Three primary patterns of inheritance in man are the following:

1) Autosomal recessive
2) Autosomal dominant
3) Sex-linked (X-chromosomal)

Autosomal Dominant Inheritance: A dominant condition is transmitted in unbroken descent from each generation to the next. Most matings will be of the form M/m x m/m (i.e., heterozygote to homozygous recessive). We would therefore expect every child of such a mating to have a 50". chance of receiving the mutant gene and thus of being affected.In this pedigree, an affected father passes the trait to half of his six children, including two daughters and a son. One of the daughters passes the same trait to one of her three children.

Autosomal Recessive Pedigree:A recessive trait will only manifest itself when homozygous. If neither parent has the characteristic phenotype (disease) displayed by the child, the trait is recessive. If it is a severe condition it is unlikely that homozygotes will live to reproduce and thus most occurrences of the condition will be in matings between two heterozvgotes (or carriers). An autosomal recessive condition may be transmitted through a long line of carriers before, by the ill chance, two carriers mate. Then there will be a one fourth chance that any child will be affected.

If the parents are related to each other, perhaps by being cousins, there is an increased risk that any gene present in a child may have two alleles identical by descent. The degree of risk that both alleles of a pair in a person are descended from the same recent common ancestor is the degree of inbreeding of the person.

Pedigree of Sex-linked Traits: The transmission of X-linked traits is in a zigzag manner. Females transmit X chromosomes to both sons and daughters. Males transmit the X chromosomes only to daughters and Y chromosomes to sons. The X-linked traits, which are recessive are preferentially seen in males, who are always homozygous for the X chromosomes. Females are heterozygous and form the "carriers" of that trait. Most X-linked traits are recessive.

A color-blind man is the father of "carrier" daughters and normal sons. Carrier daughters have a 50'N, chance to have color-blind sons and a cross between a color-blind male and carrier female can produce color-blind daughters. Hemophilia also has the same type of inheritance. Duchenne muscular dystrophy is another example of X-linked inheritance.Transmission of Y-linked Genes
Men are homozygous for Y-linked genes present on the non-homologous parts. All these genes will be expressed in all conditions. These genes are always transmitted from father to sons and never to daughters. There are no essential genes in the Y chromosomes except the locus for the maleness and fertility.

Pseudoautosomal Inheritance:
There are some homologous regions in the X chromosomes and Y chromosomes. These homologous parts pair during meiosis and may undergo crossing over. Therefore, genes in these homologous regions show inheritance similar to autosomal genes and are called pseudoautosomal inheritance. Such genes or characters are very rare.

Tag: Bio Technology, Bio Genetics, Pedigree Analysis