Recent Gains on Molten Globule - Essay Example

Introduction Biochemical research intrinsically involves proteins, and biochemists seek to understand the nature of many characteristics of proteins,including protein folding. As the field grows, many researchers are using protein folding as critical steps in biotechnological experiments and product creation. In many cases proteins may lose their tertiary and secondary structures through a process called denaturation, where the naturally occurring and functional native state of the protein is lost. Modern researchers have made numerous breakthroughs in the field of protein chemistry by developing strategic methods for the refolding of denatured proteins, which have proven useful in academics and industry. During initial production and isolation of proteins, many factors such as overproduction, solvent interactions, mechanical interference, or others may result in the denaturation of proteins. Understanding protein folding involves understanding both the energy landscape of the protein system, and refolding techniques have been significantly improve as time-resolved techniques, including neutron scattering, have been developed and perfected by researchers around the globe. The techniques involve observation of protein dynamics in order to assess the critical point of refolding, information which can lead to the development of refolding solutions (Bu et al. 2001). Neutron scattering and similar inventive techniques, such as stopped-flow florescence. In order to scale up for commercial use, protein refolding techniques must be scale invariant, compatible for a large range of proteins, simple to automated, and overall economical. Methods that rely on denaturant dilution and column-based methodology generally will meet these criteria (Middelberg 2002). The technology of refolding has grown exponentially in the past decade, and new methods must be carefully designed to facilitate the automated and rapid determination of the conditions that must be met for refolding in order to be commercially viable. It, however, remains to be seen if researchers can translate new technologies—and possibly even the discovery of a new protein state—into technology that will improve efficiency in bimolecular research industries. Before use, proteins are generally solubilised before use in high concentrations of quanidinium chloride (GdmCl) and urea (De Bernardez 1998 and Schwarz et al. 1998). Either of these two solvents may cause certain proteins to denature, and refolding involves diluting to a low concentration zone. Unfortunately, dilution causes some proteins to aggregate instead of refolding, as expected, generating additional problems in some biochemical experiments (Molecular Station, 2009). In order to prevent aggregation, a common procedure has been developed for these protocols. The method has two distinct phases. In the first phase, a detergent is added at the dilution step, which then forms a protein-detergent complex. In the second phase, cyclodextrin is added to the mixture (Middelberg 2008). Cyclodextrins are cyclic oligosaccharides with the shape of a hollow truncated cone with a hydrophilic exterior and a hydrophilic interior, which allows hydrophobic molecules to nestle into the cavity. The cyclodextrin’s unique shape and properties allow it to tightly bind itself to the detergent and aid in the stripping process of the protein (Aachmann et al. 2003). The refolded proteins can then be recovered through a variety of ultra-purification methods, which will be discussed in detail as appropriate in later sections of this study. Invention During a cell’s life, it can be thought of as a complex protein factory consisting of many individual workers, the ribosomes. The cell is a diverse environment that contains genetic material, protein production and storage facilities, and a barrier that selectively allows proteins to exit the cell. Proteins are made from mRNA that originates in the nucleus and is translated to a protein at the ribosomal site (Protein Synthesis 2010). Because the intracellular environment contains a plethora of both macromolecular compounds, small molecules, and ions in aqueous solution, a major fraction of the cellular interior is filled with compounds than may often come in contact with one another. If the volume is excluded, the configurational entropy is lowered, increasing energy and the potential of macromolecules in solution (Ellis 2001). Zimmerman and Minton predict that association constants under these particularly crowded situations are likely to have an elevated magnitude compared to similar but dilute solutions, a factor that certainly plays into the chemistry of the cell (Minton 1993). This has the ramification that cells must develop methods of dealing with aggregation, as it is more likely to occur in the concentrated intracellular environment than in dilute solution. In order to attain protein for biopharmaceutical applications or research, the protein must be expressed, normally in a vector such as e. coli, and it must then be harvested and purified before it can be of use. Misfolded or denatured proteins must be returned to their native, bioactive state, or assays that researchers complete could fail due to lack of bioactivity. In the case of biotheraputics, loss or lack of bioactivity could mean that a drug failed to have its intended activity in vivo. The previously discussed technique, in which the protein is solubilised with detergent and cyclodextrin was patented in 1996, after being submitted to the US patent office originally in 1994 (Method for Refolding 1994). This method is commercially viable for a number of reasons, the key of which are listed below as the benefits of this invention. Benefits of Invention The key benefits of the above method are: 1. The materials are simple and inexpensive 2. Overproduced proteins may be efficiently refolded, even when those proteins are resistant to other conventional refolding techniques often used in biochemical laboratories. 3. Yields of proteins are generally increased after purification 4. Optimal detergent conditions are a native protein function, and vary by protein, but are easily solved under normal laboratory conditions 5. A wide variety of different proteins including hormones, enzymes, DNA-binding proteins, phosphatases, kinases, interleukins, enzyme inhibotor proteins, and metabolite binding proteins are all amiable to this method, and show equally positive results (Method for Refolding 1994). Aggregates A protein’s bioactivity is enabled by the native three dimensional structure it achieves, which is represented by the secondary and tertiary protein structures. When aggregation occurs, the three dimensional structure is changed and bioactivity may be reduced drastically, which is why early detection of aggregation is extremely important (Cromwell et al. 2006). Steps to remove aggregation have been developed and applied on manufacturing scales in biotherapeutic industries. One leader in the biopharmaceutical industry, Borean pharma, holds multiple patents for proteins chemistry, including patent number 0686162 that explicitly details a method for folding proteins. The Aarhus-based company has also procured equivalent US patents. The method basically refolds proteins with high yields by successive denaturation steps followed by renaturation, resulting in a dramatic increase in correctly folded protein over previous methods. The method offers extremely high yields, and as suchit is ideal for difficult to attain or rare protein work. Unfortunately, it has been shown to scale up poorly, and thus is of limited commercial value (Borean pharma 2003). The work of numerous biopharmaceutical companies has provided the world with valuable method that may one day change the face of protein chemistry. Additionally, the efficiency of action of some metabolic pathways is increased by this crowding (Ellis 2001). When the volume is excluded, the configurational entropy of the cellular system is reduced, increasing the energy and potential of solutes (Young, Hugh and Freedman 2008). Macromolecular crowding acts to either destabilize or stabilize the forward or reverse reaction, and will favour the state of matter which excludes the volume of the macromolecular species present in the entire solution. As Zimmerman and Milton predict, the association constant under crowded situation is high in magnitude (Minton 2001). This implies that the aggregation of refolding protein molecules is an extremely pertinent problem that gains precedence in crowded situation over dilute ones. Molten Globule It has been largely debated whether the protien form termed molten globule is actually a third phase of protien or merely a transition state. If the molten globule is truly a third state of protien configuration, and truly stable, it could provide the basis for numeroud commercially viable biopharmacutical refolding applications. Normal protiens exhibit a first order change between the folded and unfolded state; however, some conditions, protiens can exhbit another stable state of partial order (Pande and Rokhsar 1997). The molten globule form and it equilibrium properties can be studied by using a Mone Carlo simulation, wherein the polymeric entropy of the polypeptide chain as it continues to flux is emphasized. Results suggest that the third state is actually thermodynamically favorable and local free energy is at a minimum. Based on numerous computer simulations coupled with physical arguments, researchers have established that that third state is analogous to the liquid state of a bulk system, and it closely parallels the solid, liquid, vapor phase diagram for fluid substances—with separation even fading along the critical point (Pande and Rokhsar 1997). If a true third protein state exists, it could serve as the basis for a revolution in the biopharmaceutical industry. The molton globule is characterized by being distinctly different in its structural arrangement from the native state of the protein and also distinctly different from the denatured state. The structure self-associates through the β-domain and possesses the ability to initiate oligomer formation. Recent Gains on Molten Globule One clinical example of the occurring in vivo is in Alzheimer’s patients, where amyloid plaques form in the brain. A unique aspect of this condition is that the stable core structure of the protofilament within each individual amyloid fibril is composed of primarily β-sheet structures but also some other undefined protein structure that is extremely stable (Binger et al. 2008). The nature of this unknown residual structure that appears in the amyloid regions suggests that some of the polypeptide actual remains independent of the distinctive cross-β structure, without a defined secondary structure type (Selkoe 2003). As shown in Figure 1, to the left, the purple color represents a β-sheet structure, the red represents a helical structure, and the dotted lines represent the undefined structure particular to amyloid protofilaments. Recent Gains Recombinant proteins are one of the newest developments to occur in biotechnology. These proteins are the result of expression of recombinant DNA, or DNA that has been reorder by researchers to meet some end goal or function, and may be used to generate unique pharmaceutical products, industrial enzymes, and other materials products (The Recombinant Protein Handbook 2010). The purification and growth of these proteins represents a unique development made possible because of increased understanding of the human genome and DNA manipulation. The human genome project is now complete; however, understanding base pairs alone is not enough. As researchers at the countries most brilliant x-ray beam line at Argonne National Labs state, structure is function (Brown 2001). Structural genomics is a dynamic and growing field, with the possibility in the future that biologists and chemists might one day be able to accurately and predictably translate linear genetic information into functional proteins. This lofty goal, however, must be preceded by a firm understanding of not only genetics, but also how the protein assumes its tertiary and secondary structure after translation. It is in this area that the work on protein refolding and intermediates is critical to the future manufacture of useful biosynthetic proteins. Researchers in the past decade have contributed greatly to the understanding of protein folding and intermediates, providing breakthroughs in the field of protein engineering that are certain to be surpassed in the coming decades. There is no single correct method for protein refolding, so the goal of researchers becomes to better understand trends and develop cohesive methods for refolding of diverse proteins under many different conditions (Chen et al. 2007). From the aforementioned harvesting of proteins by solubilization to the ability to produce genetically engineered cells as vectors for recombinant protein expression, the field is a dynamic one that is certain to have commercial impacts on biopharmaceutical and materials science related industries as it matures. Stopped-flow fluorescence anisotropy Stopped flow florescence has been utilized to investigate many various kinetics that are associated with the formation of protein structures. It is useful for observing many signally molecules and the dissociation of complex proteins in the human body (Wilkinson et al. 2001). The resultant findings show that association and dissociation energies are roughly equivalent. Experiments Conducted These were the experiments conducted in the process of coming up with scenario analysis of protein refolding. These experiments will help us evaluate the actual thought process that we have followed in our paper. We have conducted two experiments which will give us results. These results will be further analysed to come up with a confirmation of our already believed theory. The details of two sets of experiment that will be conducted are given below. (Preparative refolding Home, 2008) 1- In the first experiment the process was: Prepared 8 mole of urea+25m/g protein 1 mole of urea =60.06 8 mole of urea=12.012g And dissolved of protein 25m/g after that run of fluoresce with PH 7.3 2- In the second experiment the process was: Concentration of protein Urea =8 mole urea + protein 50 m/g ,100 mM Tirs ,1mM EDTA ,different PH to give final protein concentration 50 using (UV). Rationale of Experiments In protein engineering the proverbial golden goose lies in creating proteins with novel binding not found in nature, a feat that is often accomplished by mimicking many complex naturally occurring proteins. One example of a unique protein in nature is the antibody, which has an incredibly diverse range of binding methodologies, enabling it to bind many different chemical target molecules (Stewart 2010). The chemical complexity of the antibody is a result of millions of years of evolution, and is still unmatched by synthetic protein engineering. Many applications exist for the technology of engineering and duplicating proteins, but for these techniques to become commercially viable we must overcome engineering problems. Even slight changes in protein structure can result in dramatically reduced bioactivity, complete inactivity, or unfavorable activities that harm other proteins (Mariani 2004). To date, experimental techniques for folding and refolding have often placed their attention on characterization of the protein or on the intermediates that are formed along as the protein refolds in nature. Debate The commercial implications of the molten globule structure cause it to be a source of intense debate and the focus of numerous research groups’ studies. The structure is only partially folded, resembling the denatured state and the native state in different areas of the structure—and with some elements that are distinctly unique, which suggests that the structure is not simply a mere intermediate. The structure appears to be thermodynamically favourable over both the active and denatured states of the protein structure (Pande and Rokhsar 1997). These unique structures could enhance the understanding science has of structural proteomics, and perhaps create new areas of biotechnology based around the manufacture of proteins using these methods. Result Analysis of the experiment The interval plot was made on the bar chart of the above conducted experiments at different level of ph and at different confidence interval. Confidence interval when are changed for the collected data of the experiment gives us different charts at the same level of ph. The results change at a very high pace as we move from the position of ph of 7.4. The neutrality ph changes the result variation. This analysis suggests that protein molecules behave very differently in case of different ph. It is very different when the solution is acidic compared to the condition when the solution is basic. The graph above clearly suggests that the movement in the various ph level and how it is impacting the values on the other axis of the graph. The value drops very fast initially and then become stable at 0.003 around ph of 7.4. Then the values start rising again and but the steepness of the rise is very low. This suggest that molecules are very highly active at the ph level less than 7 and show high variability in concentration at that level. These molecules become less sensitive when the ph level goes above 7.4. Cluster Survey Analysis We conducted a cluster survey on the results of our experiment to see what the axis is which affect the analysis the most. The graph of the result is shown below. (the Massive Cluster Survey) Explanation of the Graph Above The data that we gathered through our experiment was very scattered and had different pH levels. It made a very sensible approach to use something called cluster survey to see at which pH level the concentration is maximum. This graph is drawn from that inference of cluster survey. The objective of grouping by cluster analysis is to identify the relative contribution of the variable in separating the goods. The result for the same is shown below in bullet points. Our analysis: The fixed factors are the value of concentration of molecules. The six variables on which the analysis is done are innovativeness, features and tools, privacy, ease of use, visual appeal, usefulness and speed. 1. The points that come out of the scattered plot are- 2. More numbers of clusters are concentrated around the ph below 7.4. 3. Clusters form are indicative of the high value of concentration at the given pH. 4. There has been shift in the concentration at a very high pace when the Ph is shifted. 5. The analysis suggests that there are very few clusters formed among the result and the result is very highly variable. (Discriminant functional Analysis, 2008) The interval plot was made on the bar chart of the above conducted experiments at different level of ph and at different confidence interval. Confidence interval when are changed for the collected data of the experiment gives us different charts at the same level of ph. The results change at a very high pace as we move from the position of ph of 7.4. The neutrality ph changes the result variation. This analysis suggests that protein molecules behave very differently in case of different ph. It is very different when the solution is acidic compared to the condition when the solution is basic. The graph above clearly suggests that the movement in the various ph level and how it is impacting the values on the other axis of the graph. The value drops very fast initially and then becomes stable at 0.003 around ph of 7.4. The values then start rising again and but the slope of the rise is very low. This suggest that molecules are very highly active at ph levels less than 7 and show high variability in concentration at that level. These molecules become less sensitive when the ph level goes above 7.4. pH 5.4 PH6 PH7 PH8 PH9 320 330 340 345 350 This graph shows the analysis of how the wavelength value will change with different levels of ph. The analysis shows that values keep on increasing as the pH level in the system is increased. This increase has been very steady and the jumps has been steady Wavelength Analysis Our next objective was to analyse the emission at different ph levels .We plotted the graph for emissions against the various ph levels to perform this analysis. Let us have a look at some of the results from the same. The confidence interval for the same is still kept as high as 95% for our data analysis. The results from the same indicate that at 95% confidence interval the value of peak height of maximum emission wave length is tending to form a normal curve skewed towards the ph of 5.4 to ph of 6.4. These implications suggest that there are very high values of peak height of maximum emission wave length at around ph of 5.4 and 6.4. The value go as high as 800 and reaches its peak at ph of 7.4 where the value is around 800. After that the value of peak height of maximum emission wave length tends to go down but the curve of the downward slope is not very steep. Ph level analysis at different Concentration   01:05 01:10 01:50 320 0.006 0.005 0.011 330 0.005 0.004 0.01 340 0.004 0.006 0.009 350 0.003 0.002 0.005 360 0.005 0.002 0.005 370 0.003 0.001 0.005 380 0.001 0.003 0.004 390 0 0.004 0.003 400 0 0.002 0.003 410 0 0.001 0.003 420 0 0.001 0.003 Average 0.002455 0.002818 0.005545 StDev 0.002339 0.001722 0.003012 The average values at different concentration level of 1:05, 1:10, 1:50 was analysed. The results of the calculation are shown in the table. The low level of standard deviation suggests that results are more in sync at the same concentration level. There is now high variation in the data. The graph for the same is shown below. Reason of Discriminant Analysis PURPOSE The main objective of discriminant analysis is a functional analysis which can predict group behaviour based on a set of linear combinations of all the available interval variables. The procedure of the analysis is to analyse a set of observations and develop a model which can suggest the data to us. This analysis is done on data which we got through our experiment to understand which function on the axis is setting the trend for the results. This data clear suggest that most of the data point are concentrated at a high value of function 2(ph levels) compared to function 1. The concentration is as high as 63.2% compared to 29.4%. (Discriminant functional Analysis, 2008) Final Comments on total Data Analysis The interval plot was made on the bar chart of the above conducted experiments at different level of ph and at different confidence interval. Confidence interval when are changed for the collected data of the experiment gives us different charts at the same level of ph. The results change at a very high pace as we move from the position of ph of 7.4. The neutrality ph changes the result variation. This analysis suggests that protein molecules behave very differently in case of different ph. It is very different when the solution is acidic compared to the condition when the solution is basic. We conducted a cluster survey on the results of our experiment to see what the axis is which affect the analysis the most. The graph of the result is shown below. The objective of grouping by Discriminant or cluster analysis is to identify the relative contribution of the variable in separating the goods. The fixed factors are the value of concentration of molecules. The six variables on which the analysis is done are innovativeness, features and tools, privacy, ease of use, visual appeal, usefulness and speed. The interval plot was made on the bar chart of the above conducted experiments at different level of ph and at different confidence interval. Confidence interval when are changed for the collected data of the experiment gives us different charts at the same level of ph. The results change at a very high pace as we move from the position of ph of 7.4. The neutrality ph changes the result variation. This analysis suggests that protein molecules behave very differently in case of different ph. It is very different when the solution is acidic compared to the condition when the solution is basic. The graph above clearly suggests that the movement in the various ph level and how it is impacting the values on the other axis of the graph. The value drops very fast initially and then become stable at 0.003 around ph of 7.4. Then the values start rising again and but the steepness of the rise is very low. This suggest that molecules are very highly active at the ph level less than 7 and show high variability in concentration at that level. These molecules become less sensitive when the ph level goes above 7.4. The average values at different concentration level of 1:05, 1:10, 1:50 was analysed. The results of the calculation are shown in the table. The low level of standard deviation suggests that results are more in sync at the same concentration level. There is now high variation in the data. The graph for the same is shown below. Literature The inside of a cell is a veritable jungle of molecules, containing many different types of compounds. These intracellular environments are crowded with a diverse set of macromolecules, causing a variety of surprising effects that include buffer effect, effects on reaction rates, and effects on equilibrium of other macromolecules (Ellis 2001). The addition of high concentrations of synthetic as well as natural macromolecules to such buffered solutions enables crowding to be mimicked in experiments that are conducted in vitro. Protein aggregation can be stimulated by crowding, which may explain the existence of certain macromolecule chaperones that serve to regulate the effects of aggregation in other macromolecules. Crowding also has positive effects on the cell, enhancing the process by which polypeptide chains collapse into functional proteins as well as the assembly of oligomeric structures. Conclusion In the future development of biochemical research, further study of how proteins form will be necessary in order to increase both the capabilities and efficiency of biotechnology enterprises, especially those concerned with the development of biotherapeutics, biopharmacuticals, and other macromolecule drug compounds. Researchers are making major headway in understanding these phenomenons by observing proteins in the denatured state, the native state, and additionally in unique states, such as the molten globule and various intermediates. These states are critical to the development of improved protocols for synthetic protein folding and refolding, in order to increase yields of both wild type and recombinant proteins used in laboratory research and used in large-scale industry. The results from the same indicate that at 95% confidence interval the value of peak height of maximum emission wave length is tending to form a normal curve skewed towards the ph of 5.4 to ph of 6.4. These implications suggest that there are very high values of peak height of maximum emission wave length at around ph of 5.4 and 6.4. The value go as high as 800 and reaches its peak at ph of 7.4 where the value is around 800. After that the value of peak height of maximum emission wave length tends to go down but the curve of the downward slope is not very steep. Our next objective was to analyse the emission at different ph levels. We plotted the graph for emissions against the various ph levels to perform this analysis. Let us have a look at some of the results from the same. The confidence interval for the same is still kept as high as 95% for our data analysis. The results from the same indicate that at 95% confidence interval the value of peak height of maximum emission wave length is tending to form a normal curve skewed towards the ph of 5.4 to ph of 6.4. These implications suggest that there are very high values of peak height of maximum emission wave length at around ph of 5.4 and 6.4. The value go as high as 800 and reaches its peak at ph of 7.4 where the value is around 800. After that the value of peak height of maximum emission wave length tends to go down but the curve of the downward slope is not very steep. The interval plot was made on the bar chart of the above conducted experiments at different level of ph and at different confidence interval. Confidence interval when are changed for the collected data of the experiment gives us different charts at the same level of ph. The results change at a very high pace as we move from the position of ph of 7.4. The neutrality ph changes the result variation. This analysis suggests that protein molecules behave very differently in case of different ph. It is very different when the solution is acidic compared to the condition when the solution is basic. The graph above clearly suggests that the movement in the various ph level and how it is impacting the values on the other axis of the graph. The value drops very fast initially and then become stable at 0.003 around ph of 7.4. Then the values start rising again and but the steepness of the rise is very low. This suggest that molecules are very highly active at the ph level less than 7 and show high variability in concentration at that level. These molecules become less sensitive when the ph level goes above 7.4. Molten globule stage is one of the most debatable stages for protein structure. If it can be proved that molten globule stage can be mapped to protein structure, then many of the problems with bio pharmaceuticals will be erased. Biotechnology industries commenced with production of proteins which are recombinant. This recombinant protein is used for developing biopharmaceuticals. The scientific development in the field of recombinant technologies has contributed not only biopharmaceuticals, It also caters to drug development, industrial enzymes and material sciences. Numbers of studies are on which discusses at various levels the structure of molten globule as third phase of protein structure. In one of the studies conducted by Vijay S. Pande and Daniel S.Rokhsar, equilibrium properties of proteins were studied under Monte Carlo simulations for protein like heteropolymers. In an unfolded state it is suggested that a distinct molten globule state is visible. Recent advances in chemical proteins have enhanced scientist understanding of all the possible intermediates that may be formed at various folding and unfolding stage. References Aachmann, F.L.; Otzen, D.E.; Larsen, K. L.; and R. Wimmer. (2003). Structural background of cyclodextrin–protein interactions. Protein Engineering, 16( 12), 905-912. Binger, K.; Phan, C.; Wilson, L.; Bailey, M.; Lawrence, L.; Schuck, P.; and Howlett, G. (2008). Apolipoprotein C-II Amyloid Fibrils Assemble via a Reversible Pathway that Includes Fibril Breaking and Rejoining. Journal of Molecular Biology 376 (4), 1116-1129. Borean Pharma Strengthens Patent Position in Protein Refolding; Grant of European Patent Provides Broad Coverage for Protein Refolding Process. (28 May 2003). PR Newswire. Brown, Evelyn. (2001). Protein Structures Solved Faster with More Detail than Ever. Fronteirs 2001. Argonne National Laboratory. Viewed 26 August 2010 http://www.anl.gov/Media_Center/Frontiers/2001/b1excell.html. Bu, Z; Cook, J; Callaway, D. J. E. (2001). Dynamic regimes and correlated structural dynamics in native and denatured alpha-lactalbuminC. Journal of Molecular Biology, 312(4), 865–873. Chen, Y.; Ding, F.; Nie, H.; Serohijos, A.; Sharma, S.; Wilcox, K; Yin, S.; and Dokholyan, N. (2007). Protein Refolding: Then and Now. Archives of Biochemistry and Biophysics. Cromwell, Mary; Hilario, Eric; and Jacobson, Fred. (2006). Protein Aggregation and Bioprocessing, AAPS Journal, 8(3), E572-E579. De Bernardez , Clark, E. (1998). Refolding of recombinant proteins. Current Opinion Biotechnol. 9, 157-163. Ellis, John. (30 September 2001). Macromolecular crowding: obvious but underappreciated. Trends in Biochemical Sciences, 26(10), 597-604. Mariani, Sarah. (2004). Engineering Proteins: Engineering Proteins. Medscape General Medicine Reviews. Viewed at http://www.medscape.com/viewarticle/472465_2 Method for Refolding Misfolded Enzymes with Detergent and Cyclodextrin. (1994). United States Patent 5563057. Middelberg, Anton. (1 October 2002). Preparative Protein Refolding. Trends in Biotechnology, 20(10), 437-443. Middelberg, A.P. (2002). Preparative protein refolding. Trends in Biotechnology, 20(10), 437-43. Minton, Allen. (1993). Macromolecular Crowding and Molecular Recognition. Journal of Molecular Recognition, 6(4), 211-214. Minton, Allen. (15 February 2001). The Influence of Macromolecular Crowding and Macromolecular Confinement on Biochemical Reactions in Physiological Media. The Journal of Biological Chemistry, 276. Molecular Station. (2009). Retrieved Aug 7th, 2010, from http://www.molecularstation.com/forum/protein-science/891-protein-refolding-technics.html Pande, V.; and Rokhsar, D. (3 October 1997). Is the molten globule a third phase of proteins? PNAS, 95(4). Protein Synthesis. (2010). Viewed 26 August 2010 http://www.accessexcellence.org/RC/VL/GG/protein_synthesis.php. The Recombinant Protein Handbook: Protein Amplification and Simple Purification. (2010). Amersham Pharmacia Biotech. Viewed 26 August 2010 http://www.biochem.uiowa.edu/donelson/Database%20items/recombinant_protein_handbook.pdf Schwarz, L. and Rudolph, R. (1998). Advances in refolding of proteins produced in E. coli. Current Opinion Biotechnol, 9, 497-501. Selkoe, Dennis. (18 December 2003).Review Article Folding Proteins in Fatal Ways. Nature, 426, 900-904. Stewart, Carleton. (2010). Antibody Binding - Powerpoint lecture slides. Purdue University, RPCI Laboratory of Flow Cytometry, Buffalo, NY. Viewed 26 August 2010 at http://www.cyto.purdue.edu/flowcyt/educate/antibody/anti.htm Wilkinson, JC; Beecham, JM; and Staros, JV. (22 February 2002) A stopped-flow fluorescence anisotropy method for measuring hormone binding and dissociation kinetics with cell-surface receptors in living cells. Journal od Receptor Signal Transduction Research, 22(1-4), 357-371. Young, Hugh; and Freedman, Roger (2008). University Physics (12th Ed. ed.). Pearson Education. Altamirano, M.M., Garcia, C., Possani, L.D., Fersht, A.R. 1999. Oxidative refolding chromatography: folding of the scorpiontoxin Cn5. Nat. Biotechnol. 17, 187–191. Hevehan, D.L., De Bernardez Clark, E., 1997. Oxidative renaturation of lysozyme at high concentrations. Biotechnol. Bioeng. 54, Schlegl, R., Tscheliessnig, A., Necina, R.,Wandl, R., Jungbauer, A.,2005b. Refolding of proteins in a CSTR. Chem. Eng. Sci. 60,5770–5780. 221–230. Pande, V. S., Grosberg, A. Yu. & Tanaka, T. (1995) Macromolecules 28, 2218–2227. Shakhnovich, E. I. & Finkelstein, A. V. (1989) Biopolymers 28, 1667–1680. Kolinski, A. & Skolnick, J. (1994) Proteins 18, 338–352. Deisenhofer, J. (1981) Biochemistry 20, 2361–2370. Rudolph, R., Fischer, S., 1990. Process for Obtaining Renatured Proteins. US patent 4,933,494. Discriminant functional Analysis. (2008). Retrieved Aug 7th, 2010, from http://www.statsoft.com/textbook/discriminant-function-analysis/ Methods of refolding Proteins. (2007). Retrieved Aug 7th, 2010, from http://www.warf.org/technologies.jsp?ipnumber=P94097US Preparative refolding Home. (2008). Retrieved Aug 7th, 2010, from http://www.ncbi.nlm.nih.gov/pubmed/12220907 Protein folding & Refolding. (2009). Retrieved Aug 7th, 2010, from http://www.fasebj.org/cgi/content/abstract/6/8/2545 Protein refolding Reagants. (2009). Retrieved Aug 7th, 2010, from http://www.athenaes.com/ProteinRefoldingReagents.php refold home. (n.d.). Retrieved August 7th, 2010, from http://refold.med.monash.edu.au/ the Massive Cluster Survey. (n.d.). Retrieved Aug 7th, 2010, from http://www.ifa.hawaii.edu/~ebeling/clusters/MACS.html Trends in Biotechnology. (2008). Retrieved Aug 7th, 2010, from http://www.cell.com/trends/biotechnology/abstract/S0167-7799(02)02047-4 Read More