|Title of Invention||
"A METHOD FOR PIECING UP THE SPINNING PROCESS AND A SPINNING POSITION OF A SPINNING MACHINE"
|Abstract||Conditions for reversible thermal unfolding of proteins and for purification of proteins from inclusion bodies are protein specific. We developed a simple, scalable procedure for identifying such conditions for any protein of interest. The procedure involves incubating the protein of interest under a variety of different buffer conditions. The samples are heated to 75°C at l°C/minute in a PCR machine, cooled to room temperature and then transferred to an ELISA plate. Irreversible denaturation is readily detected by light scattering at 405 nm. Reversible thermal unfolding is confirmed by ensuring that the protein has the expected size either by native PAGE or gel filtration chromatography or both. The procedure has been used to determine conditions for reversible thermal unfolding of the dimeric protein, Cob and for S-protein; an aggregation prone fragment of Raze A. The procedure was also adapted to screen for conditions for efficient refolding of a two domain disulfide containing protein, CD4, from inclusion bodies. We anficipate that the methodology developed in this work can be readily applied to find solution conditions for reversible, chemical unfolding for a variety of proteins and to refold proteins from inclusion bodies. This procedure should be particularly useful for structural genomics efforts where many proteins of unknown structure and function are to be expressed, purified and structurally characterized.|
|Full Text||Field of Investigation:
This invention in general relates to Bio-technology further this invention relates to protein folding and stability more particularly this inventions relates to protein refolding from inclusion bodies further this invention relates to construction and development of an novel rapid screen to identify conditions for reversable thermal unfolding of proteins. Further the invention relates to various applications to study unfolding then no dynamics and inclusion body resolubalization.
The following description traces the state of Art Technology in respect of protein refolding technology and identification of limitation of current methods of refolding proteins This invention offers solutions to overcome the limitation inherent in the present state of Art.
Present state of art:
Many recombinant proteins form insoluble aggregates known as inclusion bodies when expressed in E. coli. These aggregates can be solubilized in high concentrations of denaturants such as guanidinium hydrochloride (GdnHCl) or urea. However finding procedures for efficient refolding to give active protein after resolubilization is a major challenge in biotechnology (Georgiou & Valax, 1996). Although a number of procedures have been developed, the exact conditions for refolding are protein specific (Hofmann el al.. 1995; Zardeneta & Horowitz, 1994). Hence screening for proper refolding conditions is a tedious and difficult task. A major difficulty is that there is often no convenient and rapid assay to screen for properly folded and unaggregated protein. In specific instances, procedures have been developed to refold a particular protein. Current techniques include the use of detergents, chaperones, initiation of refolding at low temperatures followed by a temperature shift and dilution and dialysis to remove denaturant (Cole, 1996; Xie & Wetlaufer, 1996; Zardeneta & Horowitz, 1994). An overview of current approaches is given in (Misawa & Kumara, 1999).
The free energy of unfolding (AG") is a quantitafive measure of protein stability. In order to determine AG" as a function of temperature (the stability curve) (Becktel & Baase, 1987) it
is essential to know the heat capacity change (ACp) and the change in molar enthalpy (AH°) upon unfolding. Differential scanning calorimetry (DSC) is the most straightforward method of obtaining this information and with current calorimeters it is often possible to obtain accurate and reliable thermodynamic parameters from submilligram quantities of protein. A major limitation in calorimetric studies is that thennal denaturation should be reversible. In many cases, (especially for mesmeric or large proteins) this is typically not the case when thermal denaturation is carried out in aqueous solution at neutral pH. In certain cases, it has been shown that the extent of reversibility can be considerably enhanced by changing solution conditions, for example pH or buffer or in the presence of additives such as osmolytes or denaturants (Kretschmar & Jaenicke, 1999; Santoro et al., 1992). Since the conditions under which reversible folding occurs are protein specific, screening for reversible-folding conditions is often laborious and protein intensive.
Current methods for refolding proteins from inclusion bodies are protein specific. For each protein, appropriate conditions and refolding steps have to be determined. The methods are generally laborious and protein intensive and may not be easy to scale up. In addition for proteins of unknown activity and function, it is difficult to design an assay to determine that properly folded protein has been produced after refolding.
Now the invention will be described in detail with reference to the drawings accompanying this specification.
Proposed solutions (with examples):
For many proteins it is likely that there will be some solution conditions in which the protein can undergo reversible, thermal unfolding. In addition, misfolded or improperly folded proteins are more likely to aggregate than properly folded ones during the process of unfolding. Based on these two assumptions, we have developed a simple and novel procedure to identify conditions for reversible, thermal unfolding of proteins. The procedure
has been applied to study protein unfolding thermodynamics and inclusion body resolubilization. The procedure involves incubating the protein of interest under a variety of different buffer conditions with or without various additives. The incubation is carried out in a 96 well plate placed in a PCR machine. The samples are heated to 75°C at 1 degree per minute, cooled to room temperature and then transferred to an enzyme linked immunosorbant assay (ELISA) reader. Irrerversible denaturation can be readily detected by light scattering at 405 nm. Reversible themial unfolding is confirmed by ensuring that the protein has the expected size through native polyacrylamide gel electrophoresis (PAGE) or gel filtration. The procedure has been applied to three proteins, a bacterial toxin CcdB, an aggregation prone fragment on Rnase S, S protein and CD4, a disulfide containing, eukaryotic protein which contains the binding site for the HIV protein gp120.
This invention thus provides a process to develop a rapid saeen for protein refolding, comprising the steps of:
a) Incubating the selected protein under a variety of different buffer conditions with or without various additives in a PCR machine by heating to a temperature of ZSX at 1 degree per minute;
b) cooling the incubated protein to room temperature and subjecting the same to optical measurements in an enzyme linked immunosorbant assay reader;
c) detecting the irreversible denaturation by light scattering at 405nm;
d) confirming the reversible thermal unfolding either through polyacrylamide gel electrophoresis (PAGE) or gel filtration or other methods for size measurement such as dynamic light scattering.
The buffers are molecules used to maintain the pH. For a given pH range, there are specific buffers that are commonly used. For example, for pH's areound 6.0, one would use acetate or citrate buffer. For pH's around 7.0, one would use phosphate or HEPES (4-2(Hydroxyethylpiperazine-1-ethanesulfonicacid) etc.
The additives are molecules that are known to affect protein stability and folding behaviour. Commonly used additives would be denaturants (such as urea or guanidinium hydrochloride), stabilizing agents (these include such as polyols such as glycerol or osmolytes such as sarcosine or betaine). However the important thing about the screening procedure is that it uses very small quantities of protein so that are large number of conditions (buffers and additives) can be screened with a limited amount of material.
CcdB is a dimeric, bacterial toxin. Previous efforts (Dao-Thi et al., 2000) to characterize the thermal unfolding by DSC were hampered by the fact that thermal xmfolding for this protein was completely irreversible. We applied our methodology on CcdB by incubating 200|4l of CcdB at a final concentration of 150 ^g/ml under a variety of different conditions. The variables included pH and various concentrations of additives such as GdnHCl, ethylene glycol, glycerol, trehalose and sarcosine. Buffers used were citrate for pH3 and 4, HEPES for pH 5-7 and Tris for pH 8 and 8.5. All additives were prepared m 50 mM HEPES, pH 7.0. A total of 42 different conditions were examined. Individual samples were prepared in 0.5 ml microflige tubes and placed in the sample block of a PCR machine. Samples were heated to 75°C at the rate pf 60'C hr'. The temperature was maintained for about 10 minutes at 75°C and then reduced to 25°C at a rate of 180°C hr"\ Samples were then transferred to an ELISA plate and the absorbance at 405 nm was measured. Irreversible unfolding was typically accompanied by sample precipitation. Precipitation can be detected either visually or by a high value of sample absorbance relative to buffer in the plate reader. In conditions where there was no visible precipitation, (for example in the presence of 2 M GdnHCl) the additive was removed by dialysis against 10 mM HEPES, pH 7.0 or by repeated cycles of concentration and buffer dilution usmg a Microcon ultrafiltration unit.
Subsequently samples were analyzed for aggregation by 10% "Native PAGE (Goldenberg & Creighton, 1984).
The data clearly indicate sets of conditions where no visible precipitation occurs. At neutral pH no precipitation occurs in the presence of moderate concentrations of GdnHCl and high concentrations of betaine, sarcosine and glycerol. Figure 1 and Table 1 clearly demonstrate the results obtained in the ELISA plate assay. The native PAGE (Figure 2) shows that the final folded form under all these conditions is similar to that of the native, folded protein. We have also carried out Thermal Denaturation studies of CcdB by DSC under the conditions where there was no precipitation (Figure 3). The DSC scans were highly reversible and accurate thermodynamic data could be obtained.
In order to minimize sample requirements, the screen was also carried out for CcdB with 25 instead of 200 \x\ of sample. In this case after heating and cooling, samples were diluted to 100 |il (the minimum volume required in the ELISA reader) with buffer. Since the precipitates remain insoluble even after dilution, the results were identical to those seen with larger sample volumes.
The screen was carried out by incubating RNase S, S protein and RNase A at about ImM concentration under a variety of conditions and subjecting them to a screen similar to that described above. S protein is an aggregation prone fragment of RNase S which in turn is derived from RNase A by proteolytic cleavage. Previous characterization of S-protein alone has shown that it aggregates at high concentrations required for DSC ((Nadig et ciL, 1996)). By our methodology we have found conditions in which S-protein is not aggregated upon heating (Figure 1 and Table 1). Native PAGE as well as DSC has confirmed that under these conditions S-protein is reversible to thermal denaturation. (Figure 4).
The method was applied to the refolding of the first two extra cellular dorriains of human CD4 from inclusion bodies. This is a disulfide containing, eukaryotic protein which contains the binding site for the HIV protein gpl20 and serves as the cellular receptor for HIV (Kwong et al., 1998). Multidomain proteins, especially those containing multiple disulfide bonds, are known to be difficult to refold. Hence, this protein is an appropriate test case for the utility of the method. Denatured CD4 purified from inclusion bodies (Figure 5) was refolded under a total of thirty different condifions. In each case 0.5 mg/ml orCD4 in 7.4 M GdnHCl, 300 mM imidazole, pH 7 was diluted 20 fold into different final folding condifions at 25°C. The final sample volume was 100 jul. After dilution, samples were incubated at 25°C for 30 minutes. Samples were then subjected to thermal unfolding by heating to 75°C and cooling back to 25°C and assayed for aggregation in an ELISA piate reader as described above. The results from the screen suggested that at pH's of 5 and below, CD4 does not show any visible aggregation. Hence, we chose this as the best condifion for refolding of CD4. Native PAGE and gel filtration using Superdex 75 (Figure. 6A) confirmed that the protein was monomeric under these conditions. Refolding in the presence of a redox buffer of reduced and oxidized glutathione did not enhance the refolding of CD4, suggesting that the material in the inclusion bodies contained correctly formed disulfide bonds. CD and Fluorescence spectra, as well as ANS binding experiments of the refolded material carried out in PBS compared well with corresponding spectra of the positive control (Figure. 6B). The activity of the refolded protein was examined by surface plasmon resonance studies. The experiments measured the enhanced binding of gpl20 to a monoclonal antibody 17b in the presence of CD4 (Hoffman et al., 1999; Zhang et al., 1999). The presence of the CD4: gpl20 complex is clearly detectable by the increase in RU upon binding of gpl20 to immobihzed 17b in the presence of CD4 relative to binding in the absence of CD4 (Figure. 7). As a control, binding experiments were also carried out with CD4 expressed in a soluble form that had not gone into inclusion bodies. The on rate constant of approximately 1X10^ M"'s"' for gpl20 binding to 17b in the presence of refolded CD4 was virtually idenfical to that obtained with protein purified from the soluble supernatant and compared well with published values (Hoffman et al., 1999; Zhang et al., 1999). The final yield of CD4 purified and refolded from inclusion bodies is about 5 mg/litre of culture.
ELISA - Enzyme linked immunosorbant assay;
PAGE - Polyacrylamide gel electrophoresis;
DSC - Differential scanning calorimetry;
(AG") - free energy of unfolding;
(ACp) - the heat capacity change upon unfolding;
(AH°) - the change in molar enthalpy upon unfolding.
Salient Features of the Invention:
The screen described above identifies solution conditions for reversible thermal unfolding. Misfolded or unfolded proteins are generally aggregation prone and are likely to aggregate and precipitate at elevated temperatures. Hence it should be possible to use this screen to identify conditions for efficiently refolding proteins from the denatured state. An obvious and important application of this procedure would be for refolding proteins from inclusion bodies. This procedure should be particularly useful for structural genomics efforts where many proteins of unknown structure and function are to be expressed, purified and structurally characterized. Using very small amounts of protein one should be able to multiple conditions using an ELISA reader. This is a simple, rapid and scaleable procedure for identifying conditions for reversible thermal unfolding as well for solubilization of inclusion bodies.
Table 1. 96 well assay for reversible thermal unfolding. Indicated below are solution compositions for the various wells of the 96 well plate in Figure. 1.
RNase S, RNase A and S pro concentrations were about 1 mM. The following abbreviations are used in the table. GdnHCl, 'Ethylene Glycol, Proline, Mannitol, 'Betaihe, 'Sarcosine, Taurine, 'TMAO, "Sorbitol, **Trehalose,Glycerol. Buffers used at each pH are indicated in Methods.
List of Figures:
Figure 1: 96 well assay for reversible thermal unfolding. Samples were subjected to thermal unfolding and cooled to room temperature as described in Methods section. A description of solution in each well is indicated in Table 1. Irreversible denaturation can often be detected by formation of cloudy precipitates.
Figure 2: Native PAGE to confirm reversible unfolding: Samples were subjected to thermal unfolding and cooled to room temperature before PAGE. Lane 1 shows native CcdB before unfolding and the remaining lanes are after unfolding and refolding in the presence of the indicated additives. Lane 2: with 2M GdnHCl (pH3), Lane 3: with 1.2 M GdnHCl (pH 7), Lane 4: with 2 M GdnHCl (pH 7), Lane 5: 5 M Sarcosine (pH 7) and Lane 6: 4 M TMAO (pH 7).
Figure 3: (A) Representative DSC profiles for the scan (o) and rescan (•) of 25 |.iM CcdB at pH 7 at a scan rate of 90 deg hr"'. (B) DSC profiles for the scan (o) and rescan (-»■) of 25 |.iM CcdB at pH 7 in 1.5 M GdnHCl. The two-state dissociation model fit to the data is shown by the continuous line and the baseline by the dotted line. (C) Baseline subtracted DSC scans of CcdB as a function of [GdnHCl]. GdnHCl concentrations used are 2.0 M (^), 1.8 M (V), 1.5 M (■), 1.2 M (o) and LO M (•). The continuous lines represent the theoretical curves generated from the fit of two-state unfolding in conjunction with dissociation.
Figure 4: DSC profile of RNase A (A) and S-protein (B) in 50 mM Sodium acetate, pH 5. Scan (o) and rescan (continuous line) almost overlap each other.
Figure 5: Expression and purification of CD4 from inclusion bodies in BL21(DE3) cells at 30°C monitored by SDS - PAGE. Lanes 1 and 2 are the whole cell lysates before and after induction respectively, Lane 3: Soluble fraction after sonication, Lane 4: Insoluble pellet after sonication, Lane 5: Purified native CD4 and Lane 6: Purified refolded CD4.
Figure 6: (A) Gel filtration profile of native (-*■) and refolded (o) CD4 on a Superdex-75 FPLC column in 50 mM sodium acetate, pH 5.0 containing 300 mM NaCI. (B). CD spectra of the native (solid line) and refolded (dotted line) CD4 in PBS.
Figure 7: Sensorgram overlays for 62.5 nM GP120 alone (thin line) and 62.5 nM GP 120 + 62.5 nM CD4 (think line) binding to an immobilized 17b Fab surface in lOmM sodium phosphate, 150mM NaCI, pH 7.4.
It is to be noted that the basic aspect of the invention and the salient feature of the invention is clearly described so that a person in the art will appreciate various aspect of the invention.
It is also to be noted that the description is in no way intended to limit the scope of the invention and that within the scope of invention various improvements and modifications are permissible.'
1. A process to develop a rapid screen for protein refolding, comprising the steps
a) incubating the selected protein under a variety of different buffer
conditions with or without various additives in a PCR machine by
heating to a temperature of 7S0C at 1 degree per minute
b) cooling the incubated protein to room temperature and subjecting the
same to optical measurements in an enzyme linked immunosorbant
c) detecting the irreversible penetration by light scattering at 405nm;
d) confirming the reversible thermal unfolding either through polyacrylamide gel electrophoresis (PAGE) or gel filtration or other methods for size measurement such as dynamic light scattering.
2. A process as claimed in aim 1, wherein the additives are selected from
denaturants, stabilizing agents including polo’s and isolates and any other
molecules previously known to affect protein stability.
3. A process to develop a rapid screen for protein refolding substantially as
hereinbefore described and illustrated in the accompanying drawings.
|Indian Patent Application Number||26/MAS/2001|
|PG Journal Number||08/2007|
|Date of Filing||21-Dec-2001|
|Name of Patentee||MASCHINENF ABRIK RIETER AG|
|Applicant Address||KLOSTERSTRASSE 20,CH-8406 WINTERTHUR.|
|PCT International Classification Number||D01H1/115|
|PCT International Application Number||N/A|
|PCT International Filing date|