Molecular and Cell Biology (MCB) Techniques in Stress Proteins Analysis!
A. Forewords:
One of the major reasons for the attraction of recombinant DNA technology relates to gene therapy in prevention of diseases developed under stresses.
Among the causes of these diseases in human and other eukaryotes, one has been ascribed to be due to accumulation of abnormal or stress proteins in cells or tissues.
These abnormal or stress proteins are formed by over expression of genes in bio-systems when the cellular organs or tissues are exposed or given various types of physiological stresses. However, in prokaryotes imposition of stresses may accumulate abnormal proteins, which may be of importance. Both prokaryotic and eukaryotic stress proteins by proper modulation and molecular redesigning or refolding may prove to be useful in combating diseases and in developing new emerging areas of stress protein biotechnology.
Therefore, skillful detection, analysis, and purification of these specific proteins is of prime importance so that upstream bioprocessing and downstream separation can be coherently developed in the form of bioprocess integration. Many precision approaches are gradually being used in developing most modern instrumental and MCB techniques in stress protein biotechnology. Brief descriptions of the techniques that are currently being adapted are discussed below.
B. Culture Bioprocessing:
In order to trap substantial amounts of induced stress proteins during cell growth by stressor, the cell needs to be grown in a culture bioreactor of specific design depending on cell type (microbial, mammalian, or plant) and oxygen response to it. For medically important stress proteins induction in mammalian cells, the suspension culture serum bottle is used in the laboratory. However, large scale production effort of specific stress protein is yet to be reported in a systematic manner.
C. MCB Techniques in SPs:
1. PCR:
Since stress proteins are gene-induced products, the study of the structure of individual stress genes in living organisms is very important. Molecular cloning is the technique that permits such study. In microorganisms this technique depends on the replication of DNA of the plasmids and other vectors during cell division.
As the amount of DNA is very small and its detection is tedious and difficult, necessity of its amplification procedure is now well known. The procedure is based on an in vivo rather than in vitro technique. This in vivo technique is known as polymerase chain reaction (PCR). For amplifying a specific DNA segment by PCR, the adapted cycle principle has been shown in the literature.
It indicates that it is not necessary to know the nucleotide sequence of the target DNA. Heat resistant DNA polymerase has been isolated from Thermus aquaticus (Taq) and utilized most commonly in PCR technique. In eukaryotic cells the technique of directional cloning cDNA representing mRNA from control and induced cells has been used.
The cDNA libraries need to be used to produce ample amounts of DNA and RNA in subtractive hybridization for the removal of sequences present in both control and induced cells. The remaining unhybridized sequences are selectively amplified by PCR and cloned to produce the required enriched library. From this library the isolation of cDNA of specific-hsp associated mRNAs needs to be carried out since a family of heat shock genes is activated by various stimuli depending on the type of the cell.
2. Northern blotting:
In northern blot analysis, the total RNA is extracted from the tissues or cells by various methods. Generally, 4-30 mg of total RNA is subjected to agarose- formaldehyde or glyoxylic gel electrophoresis and the RNA separated are transferred to nylon or nitrocellulose membrane. To fix the RNA to the membrane, the dried membrane is either placed between two pieces of 3 MM paper, and it is generally baked for 30 mins to 2 hours at 80°C in a vacuum or conventional oven or the side of the membrane carrying the RNA is exposed to ultraviolet irradiation (254 nm).
After fixation, the membrane is pre-hybridized and then hybridized with the desired labeled cDNA probes. The membrane is washed with different concentration of salts (sodium chloride, sodium citrate [SSC] or sodium chloride, sodium dihydrogen phosphate, EDTA [SSPE] at different temperatures (60°C or 55°C) or even at room temperature. Then the autoradiograms are generally obtained after exposing the membrane to x-ray films at 70°C.
RNA is quantitated spectrophotometrically, assuming that 40 μg RNA in 1 ml gives an absorbance of 1.0 at 260 nm. Integrity of RNA preparations and consistent sample loading is verified by ethidium bromide staining of the transferred RNAs.
3. 2-D gel electrophoresis:
A very useful analytical technique for the separation of proteins is by two-dimensional polyacrylamide gel electrophoresis. Proteins are separable according to their isoelectric point by isoelectric focusing, in the first dimension; and in second dimension, it separates out according to molecular weight by polyacrylamide gel electrophoresis. 2-D gel electrophoresis is of great merit in mixed protein resolution.
In isoelectric focusing gels are made within glass tubing. Samples (protein) are added on the first dimension isoelectric focusing cylindrical gels. In a typical case, the gels are run at 300 volts for 18 hrs, and then at 800 volts for one hour. If high voltage is applied, the bands become distorted.
The sample gels are equilibrated in Farrell’s SDS sample buffer for 30 min. The second dimensional gel electrophoresis is the discontinuous SDS gel system. After the preparation of sample gels from one-dimensional gel electrophoresis, they are layered onto second dimensional SDS polyacrylamide slab gels. The gels are generally electrophoresed at 20 mA constant current per gel for 5 hrs until the dye front reaches the bottom of the gel.
If the samples are radio-labeled, the gels are then dried and exposed to x-ray films, which are processed according to standard procedure. If not, the gels are stained in 0.1% Coomassie blue in 50% methanol, 10% acetic acid for at least one hour and destained by repeated treatment with 7% acetic acid and 10% methanol.