WO2001070761A1 - Uses of alkylammonium salts in protein renaturation - Google Patents

Abstract

The recovery of at least one denatured or denatured-reduced protein comprises adding at least one alkylammonium salt the protein such that the protein refolds and regains activity.

Description

DESCRIPTION USES OF ALKYLAMMONIUM SALTS IN PROTEIN RENATURATION Technical Field

The present invention relates to the use of alkylammonium salts as refolding additives and their removal from proteins by desalting methods. The present invention also relates to a method for producing soluble catalytically active proteins at high concentrations.

Background of the Invention The present invention relates to the use of alkylammonium salts to aid in the renaturation of denatured proteins. Denatured and insoluble proteins are commonly produced by recombinant methods. However, only soluble, properly folded proteins are catalytically active.

Genetic engineering has made the production of proteins routine by using prokaryotic expression systems. (Hochuli, E.; Bannwarth, W.; Dobeli, H.; Gentz, R.; Stuber, D., Bio/Tech. 1988, 1321-1325; Georgiou, G.; Clark, E.D.B., Protein Refolding, G. Georgiou, a E.D.B.C, Ed.; American Chemical Society: Washington, D.C., 1991 ; and Marston, F.A.O., Biochem. J. 1986, 240, 1-12). Although expression in E. coli and other hosts is a convenient method for the production of large amounts of protein, the absence of the proper refolding machinery in the host may lead to non-native conformations and formation of inclusion bodies. (Armstrong, N.; Lencastre, A.D.; Gouaux, E., Protein Science 1999, 8, 1475-1483). Large amounts of proteins needed for research purposes require that the proteins to be in their native conformation. Complications arise when trying to obtain active, refolded protein from the unfolded, aggregated state.

Due to the revolutionary advances in genetic expression processes, more general renaturation strategies are required. Commonly, the renaturation of inactive protein starts with the isolation of the inclusion bodies followed by dissolution of the proteins promoted with a chemical denaturant, commonly urea or guanidine hydrochloride. Dialysis or dilution of the denaturant then initiates refolding of the protein. Unfortunately, it is during this dilution process that the protein may reform inactive aggregates that further complicate the protein isolation and purification. Therefore, it is essential to optimize the conditions that minimize the formation of aggregates during refolding. Aggregation occurs upon the exposure of the hydrophobic surfaces of a protein and this phenomenon is the major reason of the failure of protein refolding. (Georgiou, G.; Bowden, G.A., Inclusion Body Formation and the Recovery of Aggregated Recombinant Proteins; Prokop, A., Bajpai, R.K. and Ho, C.S., Ed.; McGraw-Hill, Inc.: New York, pp. 333-356 and Clark, E.D.B.; Hevehan, D.; Szela, S.; Maachupalli-Reddy, J., Biotech. Prog. 1998, 1 , 47-54). Another cause of aggregation is the reshuffling of the disulfide bonds. (Thomas, J.G.; Baneyx, F., J. Biol. Chem. 1996, 271 , 1 1 141- 1 1 147). Topologically distant, but spatially close cysteine residues typically form disulfide bonds that stabilize the active conformation of proteins. During the refolding of reduced proteins, the formation of disulfide bonds between incorrectly matched cysteine residues (intra and intermolecular) can lead to non-native structures. As a result, the misfolded protein is trapped in a non-native state that leads to aggregation.

Several techniques have recently been developed to aid in the successful refolding of proteins and diminish aggregation. Dissolution of aggregates or prevention of protein aggregation can be promoted utilizing small molecule additives. These additives reduce aggregation of the peptide segments and therefore promote protein folding. Dilution additives include detergents, amphiphiles, cyclodextrins, and polyethylene glycol (Tandon, S.; Horowitz, P., J. Biol. Chem. 1986, 261 , 15615-15618; Goldberg, M.E.; Expert-Bezancon, N.; Vuillard, L.; Rabilloud, T., Folding Des. 1996, 1 , 21-27; Karuppiah, N.; Sharma, A., Biochem. Biophys. Res. Commun. 1995, 211 , 60-66; Cleland, J.L.; Wang, D.I.C., Bio/Technol. 1990, 8, 1274-1278). One diverse group of proteins, termed "chaperones" can also aid in the proper refolding of proteins. These chaperones promote proper folding by binding to the target protein to promote proper folding and inhibiting competing aggregation of the target protein. (Gething, M.-J.; Sambrook, J., Nature 1992, 355, 33-45; Hendrick, J.P.; Hartl, F.-U., Annu. Rev. Biochem. 1993, 62, 349-384).

Also, "artificial chaperone-assisted refolding" was ₍ recently developed (Daugherty, D.L.; Rozema, D.; Hanson, P.E.; Gellman, S.H., J. Biol. Chem. 1998, 273, 33961-33971 ; Rozema, D.; Gellman, S.H. J. Am. Chem. Soc. 1995, 1 17, 2373-2374; Rozema, D.; Gellman, S.H. Biochem. 1996, 35, 15760-15771 ; Rozema, D.; Gellman, S.H. J. Biol. Chem. 1996, 271 , 3478-3487), which involves two steps: (1 ) protein capture by detergent, and (2) removal of the detergent by cyclodextrins. This two-step protocol results in high yields of correctly folded and active

( protein. While this protocol is efficient at low protein concentrations, at higher concentrations of, for example, 1 mg/mL, aggregation begins to dominate the protocol and the yield of recovered active protein decreases. (Raman, B.; Ramakrishna, T.; Rao, CM., J. Biol. Chem.

1996, 271 , 17067-17072). Therefore, there is a need for a process to obtain higher concentrations of folded protein. One common protein that has been found to renature successfully at low concentration in the presence of additives is hen egg white lysozyme (HEWL). (Maeda, Y.; Yamada, H.; Ueda, T.; Imoto, T., Protein

Engineer. 1996, 9, 461-465). The refolding pathway of HEWL is well understood. (Goldberg, M.E.; Rudolph, R.; Jaenicke, R., Biochem. 1991 , 30, 2790-2797; Fischer, B.; Sumner, I.; Goodenough, P., Arch. Biochem. Biophys. 1993, 306, 183-187). Competition between aggregation and refolding is the major obstacle in the production of active enzyme. If aggregation is decreased, refolding occurs more efficiently. Refolding becomes problematic at high protein concentrations ( > 1 mg/mL) because of the propensity for aggregation. The development of a renaturation additive that prevents aggregation and enables the recovery of active protein at high concentrations would be advantageous.

The present invention provides a method which utilizes an alkylammonium organic salt, such as ethylammonium nitrate (EAN), that prevents aggregation of denatured protein during the refolding process. This one-step method leads to high yields of active protein from chemically denatured-reduced HEWL.

Summary of the Invention The present invention describes a method for the efficient recovery or renaturation of denatured and denatured-reduced proteins using at least one alkylammonium salt as an additive in renaturation.

Exemplary alkylammonium salt compounds for use in the present invention have the following structure

-NH₃ ⁺ X-

where R is CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₃, CH₂CH₂CH₂CH₂CH₃, -CH(CH₃)₂, -CH(CH₃)CH₂CH₃, CH₂CH(CH₃)₂, and the like, and preferably any five carbon (or smaller) branched or linear chain; X is NO₃-(nitrate), SO₄ ² (sulfate), H₂PO₄-(phosphate), Cl (chloride), CH₃COO^"(acetate), Br (bromide), l (iodide), CIO₄-(perchlorate), SCN- (thiocyanate) or other suitable anionic groups.

One general aspect of the present invention is a method of renaturing, or refolding, proteins using at least one alkylammonium salt as a refolding additive. The alkyl ammonium salt prevents aggregation of the denatured protein during the refolding process. The amount of added alkyl ammonium salt ranges from about 0.5% to about 50%, and in certain embodiments from about 0.5% to about 10% and from about 1.0% to about 5.0%. The ammonium salt is added to the denatured protein in buffer to stabilize the protein against irreversible thermal denaturation. Renaturation of the protein occurs at concentrations of about 10 g to about 1.6 mg/mL.

In one particular aspect, the room temperature liquid alkylammoniumsalt, ethylammonium nitrate (EAN) enhances the recovery of denatured-reduced proteins. EAN is a clear, colorless liquid at room temperature and forms a hydrogen bonding lattice similar to that of water. EAN provides a suitable non-aqueous solvent medium for proteins. When HEWL is denatured-reduced using routine procedures and renatured using EAN as an additive, HEWL regains 75% of its activity. When HEWL is denatured and reduced in the presence of EAN, dilution results in up to 90% recovery of active protein. The refolded-active protein is separated from the molten salt by simple desalting methods. The renaturation method of the present invention is a one-step process, as compared to other refolding processes. Another advantage is that the one-step renaturation method of the present invention is useful with many types of protein.

Description of the Drawings

Fig. 1 shows a stacked plot of a DSC profile of 0.069 mM HEWL in with 5% EAN solution of 0.1 M phosphate buffer, pH 6.24, vs. buffer alone; a) Initial scan of HEWL in 0.1 M phosphate buffer solution, pH 6.24; b) Initial scan of HEWL in 5% EAN solution; c) refolding scan of HEWL in 5% EAN solution; d) refolding scan of HEWL in buffer solution. Refolding occurred after initially heating to 100°C, slowly cooling to RT, equilibrating for one hour and then heating to 100°C to examine refolding of the enzyme.

Fig. 2 illustrates trie unfolding process of a protein in the presence and absence of EAN. The native protein is thermally denatured to the unfolded state. The protein in the absence of EAN as an additive aggregates when denatured and then precipitates from the solution and does not regarding activity. In the presence of EAN, the protein does not aggregate and can refold to active protein. Fig. 3 shows a representation of the renaturation procedure for

HEWL using EAN and an additive. HEWL was denatured with 6.0 M

Gdm-HCI and 43 mM DTT in 0.100M Tris-sulfate buffer, pH 8.5.

Refolding of HEWL was achieved via (a) dilution assisted protein refolding, (b) dilution assisted protein refolding using EAN in the dilution buffer. EAN was utilized at the solvent in the denaturation step of 8.0M urea and 43 mM DTT and renaturation was achieved via (c) dilution of denatured protein in EAN.

Fig. 4 shows the fluorescence spectra of HEWL in buffer (~e-- ) and after desalting from 5% EAN ( ).

Description of the Invention

Ethylammonium nitrate is a clear, colorless room temperature liquid salt. The preparation of EAN was first described in 1929 (Sugen, S.;

Wilkens, H., J. Chem. Soc. 1929, 1 , 1291-1298), but it did not attract much interest until the 1980's. EAN is a highly associated substance with many properties similar to water. (Evans, D.F.; Chen, S.-H.; W.

Shriver, G.; Arnett, E.M., J. Am. Chem. Soc. 1981 , 103, 481-482;

Evans, D.F.; Yamauchi, A.; Roman, R.; Casassa, E.Z., J. Coll. Inter. Sci.

1982, 88, 89-96; Evans, D.F.; Kaler, E.W.; Benton, W.J., J. Phys. Chem. 1983, 87, 533-535). The present invention relates to the use of alkylammonium salts, such as EAN, as renaturation additives. Thermally denatured HEWL does not precipitate from EAN solutions after it was heated to temperatures of 100°C, while the thermally inactivated enzyme in buffer precipitated out of solution. Many traditional spectroscopic techniques, such as UV- Vis and CD spectroscopies, could not be used for examining protein structure because the EAN absorption masked the characteristic protein peaks.

Calorimetry was used to investigate the influence of additives on the stability of protein structure. Differential scanning calorimetry (DSC) was employed to examine the effect of EAN on the thermal properties of the enzyme. Fig. 1 contains a DSC thermogram of HEWL (1a) and the impact of 5% EAN on its thermal stability (1 b). When the HEWL is cooled in buffer, precipitation occurs and no refolding is evident in subsequent DSC runs (1 d). Surprisingly, addition of 5% EAN to the HEWL solution in buffer stabilizes the enzyme against irreversible thermal denaturation. Integration of the thermogram of the EAN treated HEWL solution shows approximately 87% refolding (1 c). Careful inspection of Fig. 1c shows the appearance of a small shoulder indicating that the transition is no longer two-state. The presence of this additional peak indicates that there may be a small structural change in the enzyme. While not wishing to be held to one theory, this may be due to destabilization of one of the domains of the protein structure or a slight misfolding during the cooling process. Nonetheless, the sample maintains activity. Standard activity assays of this sample showed that over 90% of the original activity was retained.

Table 1 below contains the T_m of HEWL in buffer and in 5% EAN solutions after one refolding cycle. There is a 3°C drop in the T_m of HEWL upon the addition of EAN. This shows that EAN is a denaturant. In order to confirm that EAN is a denaturant, calorimetric experiments were carried out and the T_m of HEWL was monitored as a function of EAN concentration. These experiments clearly show that EAN is acting as a denaturant.

Table 1 Differential Scanning Calorimetry of HEWL in buffer and in a 5% EAN buffer solution. The data have been fitted using the ORIGIN software (Microcal Inc.)

Sample — m (initial) ΔH (i_nitja|) -i-m(refolding) AH(refolding) buffer 74.76 ± 89.8 ± no refolding no refolding 0.01 °C 0.3kcal/mol

5% EAN 71.39 ± 87.2 ± 71.23 ± 71 .8 ±

0.02°C 0.4kcal/mol 0.02°C 0.2kcal/mol

During thermal denaturation of HEWL, the hydrophobic core of the protein is exposed, but the disulfide bonds remain intact. (Khechinashvilli, N.N.; Privalov, P.L.; Tiktopulo, E.I., ΛEBS /.eft 1973, 30, 57-60; Privalov, P.L.; Khechinashvili, N.N., J. Mol. Biol. 1974, 86, 665- 684; Griko, Y.V.; Freire, E.; Privalov, G.; Dael, H.V.; Privalov, P.L., J. Mol. Biol. 1995, 252, 447-459; Ibara-Molero, B.; Sanchez-Ruiz, J.M., Biochem. 1997, 36, 9616-9624). The intermolecular association of the hydrophobic core of proteins leads to aggregation and precipitation. An interesting feature of the EAN treatment is that no precipitation occurs even after extended heating of the HEWL solution. While not wishing to be held to one theory, the inventors herein believe that the alkyl (ethyl) portion of EAN interacts with the hydrophobic portion of the protein and protects the protein from intermolecular aggregation while the anionic or charged portion of the salt stabilizes the electrostatic interactions of its secondary structure, as shown in Fig. 2. (Kohn, W.D.; Kay, CM.; Hodges, R.S., J. Mol. Biol. 1997, 267, 1039-1043). Since precipitation is a major problem in the production of active proteins via recombinant techniques, influence of EAN on the refolding and aggregation of chemically denatured-reduced HEWL was examined.

Lysozyme contains four disulfide bonds that are important in maintaining the tertiary structure of the protein. Renaturation of large quantities of chemically denatured-reduced HEWL by dilution , is an inefficient process due to the propensity for aggregation. (Goldberg, M.E.; Rudolph, R.; Jaenicke, R., Biochem. 1991 , 30, 2790-2797; Fischer, B.; Sumner, I.; Goodenough, P., Arch. Biochem. Biophys. 1993, 306, 183-187; Sundari, C.S.; Raman, B.; Balasubramanian, D., FEBS Lett. 1999, 443, 215-219). Reduced-denatured lysozyme was prepared. (Rozema, D.; Gellman, S.H., Biochem. 1996, 35, 15760-15771 ). Renaturation of HEWL was attempted using EAN as an additive in the renaturation solution. Fig. 3 provides a detailed illustration of the procedure. The results are contained in Table 2 below.

Table 2

Chemical renaturation of HEWL using EAN as an additive during renaturation-oxidation dilution process.

SamDle^b Madditive % EAN Aggregation Activity (U/mα protein) % Activitv control³ none 0% no 1.5 ± 0.1 100 ± 10%

2 none 0% yes 0.008 ± 0.001 1.0 ± 0.1 %

3 0.05 M EAN 0.5% yes 0.25 ± 0.09 16 ± 9%

4 0.16 M EAN 1 % yes 0.34 ± 0.03 22 ± 3%

5 0.54 M EAN 5% yes 1.15 ± 0.03 75 ± 3%

6 1 .01 M EAN 11 % yes 0.33 ± 0.09 22 ± 9%

7 3.07 M EAN 33% yes 0.19 ± 0.03 12 ± 3%

8 5.09 M EAN 55% yes 0.42 ± 0.03 27 ± 3%

9 0.018 M CTAB 0% yes 0.08 ± 0.02 5 ± 2%

10 0.006 M CTAB 0% no 0.058 ± 0.008 4 ± 1 %

"Control solution was diluted with buffer only and not denatured or renatured with any of the respective solutions. Denaturation conditions involve 14μL of a solution containing 8.7 M GdmHCI, 143 mM Tris buffer (pH 8.5), 43 mM

DTT. to which 6 μ of 83.5 mg/mL HEWL stock solution was added. The solution of the denatured-reduced HEWL was diluted in a 1 :1 GSH:GSSG solution with Tris buffer (pH 8.5) and the concentration of EAN as indicated.

As the denatured-reduced HEWL was diluted with renaturation solutions containing increasing amounts of EAN, the activity of the enzyme increased, reaching a maximum activity at 75% recovery in the presence of 0.54 M EAN. Recovery of active protein declines at higher concentrations of EAN. The detergent CTAB was also used in the renaturation solution, but minimal activity was regained, as shown in Table 2. At lower concentrations the hydrophobic portion of EAN deaggregates the protein, but at high concentrations, it denatures the HEWL. The trend described above was also observed in renaturation studies of rhodanese. The effects of concentration on the detergent- assisted refolding of denatured rhodanese are discussed in Tandon, S.; Horowitz, P.M., J. Biol. Chem. 1987, 262, 4486-4491 and Zardeneta, G.; Horowitz, P.M., J. Biol. Chem. 1992, 167, 581 1-5816, which found that regardless of the type of detergent, each had a specific concentration range that provide reasonable refolding yields. When the detergents employed (CTAB, Z-3-14, Triton X-100) were used to refold denatured/reduced HEWL (Rozema, D.; Gellman, S.H., Biochem. 1996, 35, 15760-15771 ) and carbonic anhydrase (Rozema, D.; Gellman, S.H., J. Biol. Chem. 1996, 271 , 3478-3487), dilution of the protein-detergent solution did not result in renatured protein. Removal of the detergent from the protein solution was necessary for refolding to occur. Common detergents with long hydrophobic alkyl chains associate with the small protein to such an extent that their displacement upon dilution is difficult. The addition of a yS-cyclodextrin is necessary to remove the associated deterge t from the protein and enable it to refold to its native conformation. While not wishing to be held to one theory, it is likely that EAN has a lower affinity for the hydrophobic portion of the protein than detergent, so at lower concentrations it can be displaced by the protein during the refolding process. Also, this extra step needed for refolding of smaller proteins may be due to increased difficulty to for the protein to dislodge the hydrocarbon chains that are involved in the refolding process.

According to the present invention, the use of alkylammonium salts prevents aggregation, since the alkyl ammonium salt has a weak association with the protein, while simultaneously allowing the protein to refold without the need for removal from the protein solution.

Other salts were evaluated to determine whether salts other than EAN are able to assist in the renaturation of HEWL. A series of alkylammonium salts and other salts were investigated using the Hofmeister series as a guideline. (Baldwin, R.L., Biophys. J. 1996, 71 , 2056-2063; von Hippel, Ph.H.; Wong, K.-Y., Science 1964, 145, 557- 580). These results are shown in Table 3 below.

Renaturation of HEWL was conducted in the presence of other salt additives. One salt, bis(ethylammonium) nitrate (BEAS) had refolding yields of 35% at a concentration of 0.5 M BEAS.

In addition to high refolding yields obtained with the alkylammonium salts, the present invention offers another advantage. It is often desirable to conduct refolding at as high of concentration as possible in order to recover a high yield of protein. Most reported studies on protein refolding are in the range of g/mL. (von Hippel, P.H.; "Wong, K.-Y., Science 1964, 145, 557-580). Gellman examined HEWL refolding at concentrations of 1 mg/mL and recovered 57% of the activity. In comparison, a final concentration of 1 .6 mg/mL was examined and showed a recovery of 75% of the activity using EAN as the refolding additive. Table 3

Chemical renaturation of HEWL using other salts as additives and 12 mM GSH (reduced glutathione) :GSSG (oxidized glutathione) during renaturation-oxidation dilution process.

SamDle^b Madditive Aααreαation Activitv (x10⁴U/mα % Activity Drotein)

1 0.07 M NaCI yes 0.04 2%

2 0.52 M NaCI yes 0.01 1 %

3 5.09 M NaCI yes 0.04 2%

4 0.07 M KCI yes 0.03 ± 0.01 2 ± 1 %

5 0.51 M KCI yes 0.04 ± 0.01 2 ± 1 %

6 3.04 M KCI yes 0.01 ± 0.01 1 ± 1 %

7 0.05 M BEAS little 1.64 ± 0.01 92 ± 1 %

8 0.50 M BEAS little 1.47 ±0.02 82 ± 2%

9 5.01 M BEAS little 1.26 ± 0.01 70 ± 1 %

10 0.07 M EAP no 1 .59 ± 0.01 89 ± 1 %

1 1 0.51 M EAP no 1.59 ± 0.01 89 ± 1 %

12 5.06 M EAP no 1.33 ± 0.01 74 ± 1 %

^5 ^5

CM "~5 ^5 ^5 ^ 05

+1 +1 +1 r CM if) r r- σ) +1 r 00 ^ O P^ CM ^■H +1

0) 0) r— r— o

CO 00 CO ^τ— o

δ CM O o δ δ o d d 6 O d d d

+1 ^■H CM o I

CM CM if) +1 +1 +1 σ) o If) *"" *" d *" d

If) O <* δ δ σ) r^ ^ ^ d d -^

α) ω o 0) O o co o o (0 o o c c c c CO c c

CO <t Lf⁾ C0 r^ C0 0⁾ O r- CN C0 ^r τ- τ- τ- τ- - τ- τ- CM <N CM CM ol|

Lf) ^aControl solution was diluted with buffer only and not denatured or renatured with any of the respective solutions. ^bDenaturation conditions involve 14 /L of a solution containing 8.7 M

GdmHCI, 143 mM Tris buffer (pH 8.5), 43 mM DTT to which 6 μL of 83.5 mg/mL HEWL stock solution was added. The solution of the denatured-reduced HEWL was diluted with a solution of 12 mM

GSH:GSSG solution and Tris buffer (pH 8.5) and the concentration of salt as indicated.

BEAS = Bis (ethyl ammonium) sulfate. EAP = Ethyl ammonium phosphate.

PAN = propyl ammonium nitrate.

BAN = Butyl ammonium nitrate.

An alternative denaturation of HEWL was carried out in anhydrous

EAN containing DTT and urea (shown as Scheme b in Fig. 3). (Makhatadze, G.I.; Privalov, P.L., J. Mol. Biol. 1992, 226, 491 -505;

West, S.M.; Guise, A.D.; Chaudhuri, J.B., Food Bioprod. Process. 1997,

75, 50-56; Vanzi, F.; Masan, B.; Sharp, K., J. Am. Chem. Soc. 1998,

120, 10748-10753; von Hippel, P.H.; Wong, K.-Y. Science 1964, 145,

557-580). When the denatured sample was assayed without prior renaturation, no activity was present. Surprisingly, when the EAN denatured-reduced solution was diluted with the renaturation solutions, minimal precipitation was present and 90% of the activity was regained.

Similar results were obtained using another alkylammonium nitrate salt, butylammonium nitrate (BAN). These data are contained in Table 4 below. Another advantage of the present invention that EAN is easily removed from the protein solutions by simple desalting techniques. Fig.

2 indicates that removal of EAN from HEWL shows the intrinsic fluorescence spectrum of the native enzyme. Table 4

Ihemical Renaturation using EAN in the denaturation-reduction procesj

Sample Denaturation —additive Activity % Activity Conditions (U/mq

Drotein) control⁸ none none 1.5 ± 0.1 100 ± 10%

1 1^b pure EAN none 1.4 ± 0.1 90 ± 10%

12^b pure EAN 0.54 M EAN 1.46 + 0.08 95 ± 8%

13^b pure EAN no dilution 0.008 ± 1 .0 ± 0.003 0.3%

14° pure BAN 0.50 M BAN 1.41 ± 0.06 94 ± 6%

15° pure BAN none 1.32 ± 0.01 88 ± 1 % ^aControl solution was diluted with buffer only and not denatured or renatured with any of the respective solutions. Denaturation conditions involve mixing 14//L of a solution containing 8 M urea, 43 mM DTT dissolved in pure EAN to with 6 μL of 83.5 mg/mL HEWL stock solution. Solution was diluted with a 1 :1 GSH:GSSG solution with Tris buffer (pH 8.5) to yield a final concentration of 1.6 mg/mL of HEWL. For solution 13, no renaturation buffer was added. "Denaturation conditions involve mixing 14/ L of a solution containing 8 M urea, 43 mM DTT dissolved in pure BAN to with 6 μL of 83.5 mg/mL HEWL stock solution while being heated to 40°C in a water bath. Solution was diluted with a 1 :1 GSH::GSSG solution with Tris buffer (pH 8.5) to yield a final concentration of 1.6 mg/mL of HEWL.

The results of the experiments contained in Tables 2 and 3 clearly show that EAN is a useful additive for renaturation of reduced-denatured lysozyme. Similar results (88%) were obtained using another ammonium salt, butylammonium nitrate (BAN). Another advantage of present invention is that EAN is easily removed from the protein solutions by simple desalting techniques. Fig. 4 indicates that no changes in protein structure occur upon the removal of EAN from the protein solution.

Other than high refolding yields obtained with EAN, the present invention offers another advantage. When designing protein refolding experiments, it is most often desirable to conduct protein refolding at the highest concentration possible. Many studies were conducted on the range of micrograms when examining protein refolding. For example, HEWL refolding at concentrations of 1 mg/mL showed a recovery 57% of the activity. (Rozema, D.; Gellman, S.H., Biochem. 1996, 35, 15760- 15771 ). In the present invention, a final concentration of 1.6 mg/mL was examined with 75% of the activity recovered when implementing EAN as the refolding additive.

In the case of many proteins, dilution of denatured proteins not only decreases the apparent concentration of the denaturant (urea, guandidinium hydrochloride), but the dilution also allows the protein to refold by reducing the probability of intermolecular interactions between polypeptide chains. Remarkably, according to the present invention, renaturation using an alkylammonium salt in place of detergent does not require a second dilution because the presence of the alkylammonium salt in small amounts as an additive does not inhibit folding. The alkylammonium salt prevents aggregation of the protein. The presence of the alkylammonium salt clearly reduces aggregation and easy removal of the alkylammonium salt allows proper refolding of the protein to an active state.

The present invention thus offers an alternative strategy for protein renaturation using an easily prepared salt that does not perturb the structure of the protein significantly. The present invention also increases the active protein recovery and removes a step from the renaturation process. The use of the alkylammonium salt, such as EAN, as a renaturation additive to denatured-reduced HEWL is a novel approach to enhancing protein recovery. This is especially surprising since while one would suspect that this polar medium would be unsuitable for protein studies. However, the present invention shows that the unique physical characteristics of alkylammonium salts, such as EAN, make the alkylammonium salts a useful renaturation additive for protein renaturation.

Example Denaturation-Reduction of Lysozyme

A solution of 25 mg/mL lysozyme is 6 M GdmHCI, 100 mM Tris sulfate, pH 8.5, and 30 mM DTT was prepared by the addition of 6 L of 83.5 mg/mL lysozyme stock solution to 14/yL of 8.7 M GdmHCI, 143 mM Tris sulfate, and 43 mM DTT solution. Denaturation in neat EAN was carried out by adding 6 //L of HEWL stock solution to 14//L of 8 M urea and 43 mM DTT dissolved in EAN. After vortexing, the solution was allowed to sit overnight at room temperature.

Renaturation-Oxidation of Lysozyme The 25 mg/mL solution of denatured-reduced lysozyme solution was diluted with 143 mM Tris sulfate buffer (pH 8.5) containing 4 mM GSH and 4 mM GSSG with varying concentration of salt to give final lysozyme concentration of 1.6 mg/mL. Solutions were stored at room temperature for 24 hours before being assayed for enzymatic activity.

Assay of Enzymatic Activity

The assay for enzymatic activity was adopted from a standard assay from published procedures. (Jolles, P., Methods Enzymol. 1962,

5, 137-140). A stock solution of 0.3 mg/mL M. lysodeikticus cell suspension was prepared in 0.1 M phosphate buffer, pH 6.24. To a 3- mL cuvette, 2.99 mL of suspension was added, followed by 13 μL of renatured-oxidized lysozyme solution. The cuvette was inverted once and then placed in an UV-Vis spectrophotometer. The decrease in the light scattering intensity of the solution was then measured by following the decrease in apparent absorbance of the solution at 450 nm.

Differential Scanning Calorimetry Experiments Protein and reference solutions were degassed for 15 minutes before data acquisition. HEWL (0.069 mM) and the reference solution each approximately 1.5 mL in volume, were loaded into their respective cells in the MicroCal Differential Scanning Calorimeter. An external pressure of 30 psi was applied with nitrogen gas to both sample and reference cells. The sample was scanned relative to the reference solution over a temperature range of 15°-100°C at a rate of 90°C/hr. Heat capacity (ΔC_P) plots were baseline-corrected according to standard techniques. (Haynie, D.T.; Freire, E., Anal. Biochem. 1994, 216, 33-41 ; Duguid, J.G.; Bloomfield, V.A.; Bevevides, J.M.; George J. Thomas, J., Biophys. J. 1996, 71 , 3350-3360). The calorimetric enthalpy (ΔH_ca,) changes were obtained as areas under plots versus temperature of ΔC_P and ΔC_P/T, respectively.

For experiments involving refolding after thermal denaturation, solutions were slowly cooled to room temperature and then re- equilibrated for another hour. After equilibration, another scan of the same solution was performed and data were collected. These data were then fit as described above and percent refolding was obtained from comparison of enthalpies of the initial peak and refolding peak.

Desalting of HEWL solution A 5 mL solution of 0.32 mM HEWL (Sigma, Lot 16H6830) in a 50% EAN solution was placed in two Centriprep centrifugal concentrators, with a 3,000 molecular weight cut-off, and centrifuged for four hours. The solutions were washed 6 times with 0.1 M phosphate buffer, pH 6.24. After washing, the remaining solution was diluted to 5 mL and examined using UV-Vis and fluorescence spectroscopies. The UV-Vis spectrum was i monitored at 280 nm for evidence of denaturation. (Raman, B.; Ramakrishna, T.; Rao, CM., J. Biol. Chem. 1996, 271 , 17067-17072). Changes in the tryptophan fluorescence spectra of HEWL were monitored at 360 nm.

The above detailed description of the present invention is given for explanatory purposes. It will be apparent to those skilled in the art that numerous changes and modifications can be made without departing from the scope of the invention. Accordingly, the whole of the foregoing description is to be construed in an illustrative and not a limited sense, the scope of the invention being defined solely by the appended claims.

Claims

WE CLAIM: 1. A method useful for the recovery of at least one protein comprising adding at least one alkylammonium salt the protein, wherein the protein refolds and regains activity.

2. The method of claim 1 , in which the salt is used as a non- aqueous solvent medium for the protein.

3. The method of claim 1 , in which the alkyl ammonium salt comprises

R NH₃ ⁺ X-

R is CH₃, CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₃, CH₂CH₂CH₂CH₂CH₃, -CH(CH₃)₂, -CH(CH₃)CH₂CH₃, CH₂CH(CH₃)₂, or any five carbon (or smaller) branched or linear chain; X is NO₃-(nitrate), SO₄ ² (sulfate), H₂PO₄- (phosphate), C.^'(chloride), CH₃COO^"(acetate), Br (bromide), l (iodide), CIO₄-(perchlorate), SCN (thiocyanate) or other suitable anionic groups.

4. The method of claim 1 , in which the refolded protein is separated from the salt using a desalting process.

5. The method of claim 1 , in which the alkylammonium salt prevents aggregation of the protein during the refolding process.

6. The method of claim 1 , in from about 0.5% to about 10% alkylammonium salt is added to the denatured protein in buffer to stabilize the protein against irreversible thermal denaturation.

7. The method of claim 6, in which the amount of salt ranges from about 1 .0 to about 50.0%.

8. The method of claim 6, in which the amount of salt ranges from about 1.0 to about 5.0%

9. The method of claim 1 , in which the protein shows at least about an 75% regain of activity.

10. The method of claim 3, in which the alkyl ammonium salt comprises ethyl ammonium nitrate (EAN).

1 1. The method of claim 3, in which the alkyl ammonium salt comprises butyl ammonium nitrate (BAN).

12. The method of claim 3, in which the alkylammonium salt comprises bis(ethlyammonium) sulfate (BEAS).

13. The method of claim 3, in which the alkylammonium salt comprises ethyl ammonium phosphate (EAP).

14. The method of claim 3, in which the alkylammonium salt comprises propyl ammonium nitrate (PAN).

15. The method of claim 1 , in which the protein comprises a denatured protein.

16. The method of claim 1 , in which the protein comprises a denatured-reduced protein.

17. The method of claim 1 , in which the protein comprises hen egg white lysozyme.

18. The method of claim 1 , in which renaturation of the protein occurs at concentrations of about 10 μg to about 1.6 mg/mL.