Biotechnology : Membrane Protein

Describe about the Biotechnology for Membrane Protein.

 

1. As the G-coupled protein receptor are also an important class of membrane protein. Therefore the approach and technique that should be taken to determine the molecular structure of a particular G-coupled protein receptor is done by comparing the experimental mass date of the G-coupled protein receptor with the theoretical mass data that is generated from the previously predicted protein sequence. The homology comparison can very useful in this approach because it can identify the changes in the modification and the expression of the protein under different conditions. The techniques used for this approaches is at first the protein is separated by the process of 2D gel electrophoresis (Schaal et al. 2012). The 2D chromatography like the ion exchange chromatography and reverse phase high performance liquid chromatography (HPLC) can be more effective than the 2D gel in some cases such as the low abundance or hydrophobic proteins. Then the protein spot is to be cut from the gel and then the protein is digested by different protease enzyme like trypsin. This digestion will generate a unique fragment pattern or finger print. After this mass spectroscopy is used for the analysis of this fragment. The mass spectroscopy will generate a protein specific “mass finger print”. To identify the protein the “mass finger print” of the protein which is tested is then matched with the predicted “mass finger print” of several proteins that are present in the protein sequence data bases (Sugimoto et al. 2012 pp.96-108). For this matching process many web based programs are available which assist the searching procedure of the sequence databases with the data of the mass spectrometry. The web based available programs that are present online are MS-Fit, Mascot ms/ms and Mascot PMF. The reason behind using the mass spectroscopy for the determining the molecular structure of the protein is that the results of the mass spectroscopy is very sensitive and accurate. The result of the mass spectroscopy is also very fast, these are the reason that mass spectroscopy has become the indispensable technique for the analysis of the biomolecules.

 

Figure 1 Graph of Gel Electrophoresis

Source: (Google.co.in, 2016)

2. The hormones are one type of protein and so if the researcher have to find which hormones are involved in causing the particular type of prostate cancer. The researcher have to understand the protein- protein interaction. To study protein-protein interaction several biophysical, genetic and biochemical experimental methods have been developed. For the understanding the protein-protein interaction that researcher can utilize is Fluorescence resonance energy transfer (FRET). In this technique the two hormone proteins are to tag one with the Cyan fluorophore protein (CFP) and the other with the yellow fluorophore protein (YFP). The CFP is excited light of wavelength of 436nm and if the protein- protein interaction does not occur then the excitation of the CFP results in the emission of the light at a wavelength of 475nm on the other hand if the protein- protein interaction occurs perfectly then the CFP and the YFP are brought into close proximity. This leads to the energy transfer from the CFP to the nearby YFP. This energy transfer is referred to as FRET. The light emission from the YFP can be detected at a wavelength of 528nm. In this process it will become evident that whether this protein-protein interaction is occurring in the normal cells or in the cancer cells just by comparing their activities (Yuan et al. 2013 pp.1462-1473).

 

 

Figure 2 Fluorescence resonance energy transfer (FRET)

Source: (De Baerdemaeker et al. 2013)

 

Figure 3 Graph of FRET

Source: (Google.co.in, 2016)

3. As the ATP synthase of the novel organism is functional and it has essential subunits that are homologous to only 5 essential subunits out of the 8 essential subunits present in the ATP synthase of the all the studied organisms. These subunits are essential for several complex functions. From these findings it can be concluded that the 3 different subunits present in the ATP synthase of the novel organism possess the ability to fulfil all the functional roles of the missing essential subunits. To identify the novel subunits first the proteins of the subunits of both the novel organism and the other studied organisms are to be isolated with the help of gel electrophoresis (Jordan and Dalmasso, 2015). Then taking proteins of the subunits that are similar to the missing essential subunits are digested with the help of the protease enzyme. This type of digestion will generate a unique fragment pattern which will act as finger print which is analysed by mass spectroscopy (Adams, 2012). The “mass finger print” generated due to mass spectroscopy is then used to find homology between the proteins of the novel organism and the proteins present in the protein sequence data base. To perform this homology identification different web based programs like Mascot ms/ms, MS-Fit and Mascot PMF can be used.

 

 

Figure 4 Graph of Mass spectroscopy

Source: (Google.co.in, 2016)

Reference

Adams, R.P., 2012. Identification of essential oils by ion trap mass spectroscopy. Academic Press.

De Baerdemaeker, T., Lemmens, B., Dotremont, C., Fret, J., Roef, L., Goiris, K. and Diels, L., 2013. Benchmark study on algae harvesting with backwashable submerged flat panel membranes. Bioresource technology, 129, pp.582-591.

Google.co.in. (2016). Images of graph of FRET - Google Search. [online] Available at: https://www.google.co.in/search?q=Images+of+graph+of+FRET&espv=2&tbm=isch&imgil=gU7Xvuc-iqk6ZM%253A%253Bnlcc0TO9FWSlXM%253Bhttp%25253A%25252F%25252Fwww.semrock.com%25252Ffret.aspx&source=iu&pf=m&fir=gU7Xvuc-iqk6ZM%253A%252Cnlcc0TO9FWSlXM%252C_&usg=__UN4X3UDAeFIZXoPKNuLKluWjuhs%3D&biw=1366&bih=613&ved=0ahUKEwi-z8KIpPXPAhWIPI8KHX1SB14QyjcIOQ&ei=ePUOWL6zCYj5vAT9pJ3wBQ#imgrc=gU7Xvuc-iqk6ZM%3A [Accessed 25 Oct. 2016].

Google.co.in. (2016). images of graphs of gelelectrophoresis - Google Search. [online] Available at: https://www.google.co.in/search?q=images+of+graphs+of+gelelectrophoresis&espv=2&tbm=isch&imgil=DeqUs2WJ0lgviM%253A%253Bny3QtfrsQkL2NM%253Bhttps%25253A%25252F%25252Focw.mit.edu%25252Fcourses%25252Fbiological-engineering%25252F20-109-laboratory-fundamentals-in-biological-engineering-fall-2007%25252Flabs%25252Fmod1_2&source=iu&pf=m&fir=DeqUs2WJ0lgviM%253A%252Cny3QtfrsQkL2NM%252C_&usg=__sGH4UamU30NFaGaqYHsx6MowtJk%3D&biw=1366&bih=613&ved=0ahUKEwjDrfzPn_XPAhXIRY8KHWH3AGEQyjcINg&ei=z_AOWIOKMsiLvQTh7oOIBg#imgrc=DeqUs2WJ0lgviM%3A [Accessed 25 Oct. 2016].

Google.co.in. (2016). images of mass spectroscopy graph of 8 proteins - Google Search. [online] Available at: https://www.google.co.in/search?espv=2&biw=1366&bih=613&tbm=isch&sa=1&q=images+of+mass+spectroscopy+graph+of+8+proteins&oq=images+of+mass+spectroscopy+graph+of+8+proteins&gs_l=img.3...436785.441114.0.441465.0.0.0.0.0.0.0.0..0.0....0...1c.1.64.img..0.0.0.yHwvTAXUdt8 [Accessed 25 Oct. 2016].

Jordan, K. and Dalmasso, M., 2015. Pulse field gel electrophoresis. Methods in Molecular Biology, 1301.

Schaal, B.A. and Anderson, W.W., 2012. 74-3 An outline of techniques for starch gel electrophoresis of enzymes from the American Oyster Crassostrea virginica Gmelin.

Sugimoto, M., Kawakami, M., Robert, M., Soga, T. and Tomita, M., 2012. Bioinformatics tools for mass spectroscopy-based metabolomic data processing and analysis. Current bioinformatics, 7(1), pp.96-108.

Yuan, L., Lin, W., Zheng, K. and Zhu, S., 2013. FRET-based small-molecule fluorescent probes: rational design and bioimaging applications. Accounts of chemical research, 46(7), pp.1462-1473.