Method of solubilizing, purifying, and refolding protein

Details Method of solubilizing, purifying, and refolding protein Method of solubilizing, purifying, and refolding protein

1999-06-11

2001-11-27

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Separation or purification

C530S351000, C530S412000, C530S416000, C530S427000, C530S380000, C530S411000, C530S405000, C530S350000, C435S069100, C435S196000

Reexamination Certificate

active

06323326

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The invention relates to methods useful for refolding, solubilizing, formulating and purifying proteins. These methods are particularly useful for proteins that have been engineered by genetic recombination and produced in bacterial, yeast or other cells in a form that has a non-native tertiary structure.
BACKGROUND OF THE INVENTION
To understand fully the entire process of gene expression, it is as important to understand the process for the folding of the peptide chain into a biologically active protein as it is important to understand the synthesis of the primary sequence. The biological activities of proteins depend not only on their amino acid sequences but also on the discrete conformations of the proteins concerned, and slight disturbances to the conformational integrity of a protein can destroy its activity. Tsou et al. (1988)
Biochemistry
27:1809-1812.
Under the proper conditions, the in vitro refolding of purified, denatured proteins to achieve the native secondary and tertiary structure is a spontaneous process. To avoid formation of stable, but undesired, structures, it is necessary to use the tertiary interactions (which are formed late in folding) with their high degree of selectivity power to select and further stabilize those early local structures that are on the correct folding pathway. Thus, the finite, but very low, stability of local structures could be the kinetic “proofreading” mechanism of protein folding. The activated state of folding with the highest energy is a distorted form of the native protein, and the slowest, rate-limiting step of unfolding and refolding appears to be close to the native state in terms of ordered structure. In addition, the refolding of many proteins is not completely reversible in vitro, and reactivation yields of less than 100% are frequently observed, which holds true in particular for experiments at high protein concentration, and competing aggregation of unfolded or partially refolded protein molecules may be the major reason for a lowered reversibility, as described in Fischer and Schmid, (1990)
Biochemistry
29:2205-2212.
In the case of sufficiently large protein molecules, the nascent polypeptide chain acquires its native three-dimensional structure by the modular assembly of micro-domains. Variables including temperature, and cosolvents such as polyols, urea, and guanidinium chloride, have been tested to determine their role in stabilizing and destabilizing protein conformations. The action of cosolvents may be the result of direct binding or the alterations of the physical properties of water, as described in Jaenicke et al. (1991)
Biochemistry
30 (13):3147-3161.
Experimental observations of how unfolded proteins refold to their native three-dimensional structures contrast with many popular theories of protein folding mechanisms. Under conditions which allow for refolding, unfolded protein molecules rapidly equilibrate between different conformations prior to complete refolding. The rapid prefolding equilibrium favors certain compact conformations that have somewhat lower free energies than the other unfolded conformations. The rate-limiting step occurs late in the pathway and involves a high-energy, distorted form of the native conformation. There appears to be a single transition through which essentially all molecules fold, as described in Creighton et al. (1988)
Proc. Nat. Acad. Sci. USA
85:5082-5086.
Various methods of refolding of purified, recombinantly produced proteins have been used. For example, the protease encoded by the human immunodeficiency virus type I (HIV-I) can be produced in
Escherichia coli
, yielding inclusion bodies harboring the recombinant HIV-I protease as described by Hui et al. (1993)
J. Prot. Chem.
12:323-327. The purified HIV-I protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. It was found that a higher specific activity of protease was obtained if the purified protein was dissolved at approximately 2 mg/ml in 50% acetic acid followed by dilution with 25 volumes of cold 0.1 M sodium acetate, pH 5.5, containing 5% ethylene glycol and 10% glycerol. Exclusion of glycerol and ethylene glycol led to gradual loss of protein due to precipitation. About 85 mg of correctly folded HIV-I protease per liter of
E. coli
cell culture was obtained by this method, and the enzyme had a high specific activity.
Another example of refolding a recombinant protein is the isolation and refolding of H-ras from inclusion bodies of
E. coli
as described by DeLoskey et al., (1994)
Arch. Biochem. and Biophys.
311:72-78. In this study, protein concentration, temperature, and the presence of 10% glycerol were varied during refolding. The yield of correctly folded H-ras was highest when the protein was refolded at concentrations less than or equal 0.1 mg/ml and was independent of the presence of 10% glycerol. The yield was slightly higher at 4° than at 25° C.
The refolding of Tissue Factor Pathway Inhibitor (also known variously as Lipoprotein-Associated Coagulation Inhibitor (LACI), Extrinsic Pathway Inhibitor (EPI) and Tissue Factor Inhibitor (EFI) and hereinafter referred to as “TFPI”) produced in a bacterial expression system has been described by Gustafson et al., (1994)
Protein Expression and Purification
5: 233-241. In this study, high level expression of TFPI in recombinant
E. coli
resulted in the accumulation of TFPI in inclusion bodies. Active protein was produced by solubilization of the inclusion bodies in 8M urea, and purification of the full-length molecule was achieved by cation exchange chromatography and renaturation in 6M urea. The refolded mixture was then fractionated to yield a purified nonglycosylated TFPI possessing in vitro biological activity as measure in the Prothombin clotting time assay comparable to TFPI purified from mammalian cells.
A non-glycosylated form of TFPI has also been produced and isolated from
Escherichia coli
(
E. coli
) cells as disclosed in U.S. Pat. No. 5,212,091, the disclosure of which is herein incorporated by reference. The invention described in U.S. Pat. No. 5,212,091 subjected the inclusion bodies containing TFPI to sulfitolysis to form TFPI-S-sulfonate, purified TFPI-S-sulfonate by anion exchange chromatography, refolded TFPI-S-sulfonate by disulfide exchange using cysteine and purified active TFPI by cation exchange chromatography. The form of TFPI described in U.S. Pat. No. 5,212,091 has been shown to be active in the inhibition of bovine factor Xa and in the inhibition of human tissue factor-induced coagulation in plasma. In some assays, the
E. coli
-produced TFPI has been shown to be more active than native TFPI derived from SK hepatoma cells. However, TFPI produced in
E. coli
cells is modified in ways that increase heterogeneity of the protein.
A need exists in the art of refolding recombinantly produced proteins to increase the amount of correctly folded TFPI during the refolding process. A need also exists for increasing the solubility of TFPI. Presently the yields of recombinantly produced TFPI have been lower than desirable, and a need exists in the art of producing correctly folded TFPI. See for example Gustafuson et al. (1994)
Protein Expression and Purification
5:233-241.
TFPI inhibits the coagulation cascade in at least two ways: preventing formation of factor VIIa/tissue factor complex and by binding to the active site of factor Xa. The primary sequence of TFPI, deduced from cDNA sequence, indicates that the protein contains three Kunitz-type enzyme inhibitor domains. The first of these domains is required for the inhibition of the factor VIIa/tissue factor complex. The second Kunitz-type domain is needed for the inhibition of factor Xa. The function of the third Kunitz-type domain is unknown. TFPI has no known enzymatic activity and is thought to inhibit its protease targets in a stoichiometric manner; namely, binding of one TFPI Kunitz-type domain to the active site of one protease molecule. The carboxy

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