Ultra-Purification Methods of Refolded Proteins Recovery - Research Paper Example

In order to explore the three dimensional structure of proteins, a combination of stopped-flow fluorescence anisotropy and kinetic techniques may be used. These techniques allow for experimental observation of the change in structure of [protien name] as the pH of solution changes. Analysis yield pertinent data suggesting that the native state, the denatured state, and other unique states, such as the molten globule form, occur as a predictable function of pH, information which can be applied to a number of commercial procedures. 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 inhibitor 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 such it 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. As seen in Figure 1, protien structure is critical to the binding of many molecules that allow the protein to complte its cellular activities, and other conformations likely will lose come or all activity. 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 that is critical to understanding its function. 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 One 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 by many researchers to complete investigations involving many types of kinetics that are commonly associated with the formation of protein three dimensional structures. This technique is especially useful for observing complex interactions, such as signing molecules, though it may also be applied to study of processes in the human body, such as the dissociation of naturally occurring proteins that are critical to various metabolic processes (Wilkinson et al. 2001). The experimental results of this study completed in accordance with this method suggest that association and dissociation energies are very similar, to the extent that they may be approximated at equivalent to a high degree of certainty. Experimental Experiments Conducted In order to complete an analysis of protein refolding in [Protein name], this experiment was conducted. The results allow for further analysis and development of a scenario for protein refolding. The goal of the experiment is to encourage further study and reflective thought of the process of refolding as discussed above and in outside materials pertaining to this study. Two experiments have been conducted in order to provide kinetic information on protein refolding. The results of these two methods have been further analyzed to provide confirmation to the groups theories regarding how this protein undergoes refolding. The details for each experiment are outlined below based on the procedure published by Anton Middelberg (2002): Experiment 1 1. Prepare 8 mole of urea containing 25 m/g protein 1 mole of urea (60.06) 8 mole of urea (12.012 g) 2. Dissolve 25 m/g of protein in urea 3. At a pH of 7.3 run fluoresce on mixture Experiment 2 – Protein Concentration 1. Create Urea Mixture (8 mole urea + protein 50 m/g , 100 mM Tirs , 1mM EDTA) 2. Run at different pH values in order to yield the final protein concentration 50 (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. Results : Experimental Analysis The data from the above experiments, by pH value and confidence interval, was used to construct an interval plot. When the confidence interval is changed for the collected data, the result is different charts at the same level of pH. As the pH shifts away from the 7.4, the neutral pH, the results change dramatically. The exemplifies that proteins behave very differently in situations where the pH is different, often specifically requiring a certain set of pH requirements to undergo structural changes, such as folding, unfolding, or refolding. Thus the acidity or basicity of the solution is critical to understanding how a protein assume a three dimensional structure, and manipulating that structure on either a bench or commercial scale. As indicated in Figure 1, the pH shift causes a large change as pH moves from 5.4 to 9.4. The value initially drops as the solution becomes increasingly basic, and stabilizes at approximately 0.003 A. A slight increase is seen as the solution becomes strongly basic, however the slope of the change is very small, and can be approximated as zero for our purposes. The findings suggest that at a pH value of 7 or lower, the molecules are extremely active. Because the molecules prefer an acidic environment, it is likely that these proteins occur naturally in environments that are more acidic, and lose some or all functionality when exposed to basic solutions in vivo. At pH values that are near neutral, a high variability in concentration is also seen in the sample. Sensitivity is reduces when the pH begins to become more basic than 7.4. Stopped-flow Fluorescence Anisotropy Using the freely available application provided virtually by the University of Hawaii, a cluster survey was completed in order to statistically show the natural groupings of the experimental results (The Massive Cluster Survey, 2010). The axis shown in Figure 4 represents the area of the most activity according to the analyisis performed. Explanation of the Graph Above Initial impression of experimental results suggest that the data is scattered, with some correlation by pH level. Cluster survey methods are commonly used when it is necessary to differentiate between naturally occurring groups or elements within a dataset. This method was used in order to determine which group (in the case of the study the group represents a pH level) represents the maximum concentration value. The graph in Figure 4 is the result of that study, and results are inferred below. By grouping a dataset into clusters, it is possible to determine the relative contribution of individual variables. The results are further analyzed below. Analysis of Cluster Data In order to interpret cluster data accurately, the fixed factors represent the concentration of molecules in the cluster (Discriminant functional Analysis, 2008). Using this method, several conclusions can be drawn from the data. 1. pH values below 7.4 contain more clusters 2. Clusters indicate elevated concentration values 3. Shifts in pH dramatically affect the magnitude and direction of concentration values 4. A limited amount of individual clusters can be distinguished because of the high variability of the results Data from the cluster analysis has allowed for the data to be distinguished in five groups, based on pH value (± 0.49). These groups are shown in Figure 5. Clusters by pH pH 5.4 pH 6.0 pH 7.0 pH 8.0 pH 9.0 320 330 340 345 350 Figure 5: Cluster Groups based on a pH value group ±0.49 Wavelength Analysis The correlation of wavelength to pH value can be seen in Figure 6. This graph shows the analysis demonstrates how the wavelength value will change as the pH level is varied. The values for wavelength increase linearly with increase in pH of the system. In increase is steady and with few jumps, predicting a linear relationship. Analysis of data from the emission spectra by varying pH level was completed and plotted against the various ph levels in order to graphically demonstrate the results of this procedure. Results of this analysis maintain a 95% confidence interval. The value for the peak height of emission wavelength forms a normal curved that skews towards the pH value of 5.4 to 6.4, suggesting that this dataset represents high amounts of maximum wavelength emission at these values of pH. The peak emission value is approximately 800 and occurs near 7.4, very near bodily pH. The value of the peak height of maximum emission wavelength is a descending curve with a very small negative slope. Analysis of pH Level by 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 Figure 7: pH Analysis by Concentration with Average and Standard Deviation shown The average values at different concentration levels of 1:05, 1:10, and 1:50 were analyzed, resulting in the figures shown in Figure 8. The low standard deviation suggests that when grouped by concentration levels, the results are more significant. There is a high variability in the dataset, as visually represented in the graph shown in Figure 8. Figure 8 clearly indicates that results vary dramatically by concentration value, and the largest correlating absorbance is shown when the concentration reach as value of 1:50. Grouping this dataset by concentration proved useful in further analysis. Rationale for Discriminant Analysis Purpose of Method In order to provide meaningful analysis of the data, a method called discriminant analysis was utilized. The objective of this method is to create a functional analysis with the capability of predicting group behaviour based on the linear behaviour exhibited by a group of interval variables. It allows for a better understanding of which function on the axis is responsible for generating the trends observed experimentally (Discriminant Functional Analysis, 2008). This allows for the development of a model for group activity, such as that of protein refolding, that occurs in accordance with experiment observations. The data utilized for this analysis was obtained experimentally. From the data collected in the experiment is possible to make the observation that the majority of the dataset is concentrated at high values of function 2 (representing pH levels), compared with function 1. In this experiment, the resulting concentrations are as high as 63.2% compared with only 29.4% seen previously. Final Comments on Total Data Analysis The bar chart of the analysis, seen in preceding sections of this report, was used to make an interval plot at varying levels of pH and at distinct confidence intervals. When the confidence levels are changed, the result is different charts within the same pH level. The results change drastically with pH as pH values vary from 7.4, the neutral pH value. This analysis proposed that protein molecules behave distinctly differently with varying levels of pH, suggesting that acidity and basicity is extremely important in protein dynamic. A cluster survey on the results yielded useful information about which axis was the most affected. In the analysis the fixed factors represent the concentration of molecules. The variables of analysis include pH and concentration. Subsequent discriminant analysis revealed the relative contribution of the variable in separation of the results. It has been shown that fluorescence antsotrophy is a valuable tool for measuring structural changes in proteins, including the molten globule. Though the fluorescence intensity may not change perceptibly, the fluorescence antsotrophy shows the conformational change that occurs between the initial denatured state as it moves to the molten globule state along with the causative shift in pH value (Canet 2001). The results shown here provide evidence that the molten globule does indeed form between the denatured and native states, an important biophysical transition in protien structure. Fluorescence antsotrophy is a valuable tool for probing the molten globule form, that provides invaluable information about the rotational motility of the fluorophore no provided in simple analysis of intensity. 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 The discovery of new and effective methods of manipulating proteins is crucial to science, and further study is both academically and commercial beneficial to biotechnology as a field of study and an industry. In the future, further studies of how proteins assume and lose their three dimensional structure will be necessary to increase the capabilities and efficiency of many processes in the industry, such as biotheraputics, biopharmaceuticals, and many other forms of applied macromolecular work. Contemporary researchers observe the formation of proteins in the natural state, the denatured state, various intermediates, and uniquely stable forms such as molten globules in order to better understand how protein structural dynamics take place. Understanding the conditions at which these states occur is critical to improving currently used protocols for protein folding and refolding, which will increase the efficiency of working with these compounds by increasing yields of wild-type as well as recombinant proteins. The solutions of the future will be practical in a laboratory setting, but also have the scalability necessary to be of use in industry. The results from this experiment show that at 95% confidence interval the value of the peak height of maximum emission wavelength forms a normal curve that gradually increases towards the pH values of 5.4 to 6.4, attaining peak values at a pH of 7.4—likely the natural pH at which the protein functions in vivo. After attaining this value, the curve decreases with increasing pH at a low rate. The interval plot included in this analysis was made based on a bar chart containing experimental data by pH level at varying confidence intervals, showing similar deviation as pH values are increased or decreased from 7.4. This demonstrated the sensitivity of protein macromolecules to pH conditions, and highlights the importance of controlling pH when completing experiments or protocols using these compounds, as pH may alter the three dimensional conformation, and thus the bioactivity of a compound. It has been long accepted that intermediates form between the native state and the denatured state, though no one particular intermediate has been thought to attain stability. The molten globule stage is a source of debate in modern protein chemistry because if it can be proven as a stable state between but independent of the native and denatured state, this could serve as the basis from a revolution in the biopharmaceutical industry, opening the door to innumerable possibilities for development in the field. This could significantly improve the manner in which recombinant, as well as wild type, proteins are handled in both laboratory and commercial settings, and perhaps allow for the wide-scale implementation of new chemical techniques for working with proteins that would allow macromolecular biotherapeutics to be more widely available at a lower cost. 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. 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. 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. Canet, D; Doering, K.; Dobson, C.M.; and Dupont, Y. (April 2001). High-sensitivity fluorescence anisotropy detection of protein-folding events: application to alpha-lactalbumin. Biophysics Journal, 80, 4, p.1996-2003. 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. Deisenhofer, J. (1981) Biochemistry 20, 2361–2370. Discriminant Functional Analysis. (2008). Retrieved Aug 7th, 2010, from http://www.statsoft.com/textbook/discriminant-function-analysis/ Ellis, John. (30 September 2001). Macromolecular crowding: obvious but underappreciated. Trends in Biochemical Sciences, 26(10), 597-604. Hevehan, D.L., De Bernardez Clark, E., 1997. Oxidative renaturation of lysozyme at high concentrations. Biotechnol. Bioeng. 54. Kolinski, A. & Skolnick, J. (1994) Proteins 18, 338–352. Mariani, Sarah. (2004). Engineering Proteins: Engineering Proteins. Medscape General Medicine Reviews. Viewed at http://www.medscape.com/viewarticle/472465_2 The Massive Cluster Survey. (2010). Retrieved Aug 7th, 2010, from http://www.ifa.hawaii.edu/~ebeling/clusters/MACS.html Methods of refolding Proteins. (2007). Retrieved Aug 7th, 2010, from http://www.warf.org/technologies.jsp?ipnumber=P94097US 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. 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). Pande, V. S., Grosberg, A. Yu. & Tanaka, T. (1995) Macromolecules 28, 2218–2227. 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 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 Refold. (n.d.). Retrieved August 7th, 2010, from http://refold.med.monash.edu.au/ Rudolph, R., Fischer, S., 1990. Process for Obtaining Renatured Proteins. US patent 4,933,494. 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. 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. Shakhnovich, E. I. & Finkelstein, A. V. (1989) Biopolymers 28, 1667–1680. 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 Trends in Biotechnology. (2008). Retrieved Aug 7th, 2010, from http://www.cell.com/trends/biotechnology/abstract/S0167-7799(02)02047-4 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. Read More