Chemokine compositions

Details Chemokine compositions Chemokine compositions

1998-12-18

2003-10-14

Eyler, Yvonne (Department: 1646)

Drug, bio-affecting and body treating compositions

Lymphokine

C530S300000, C530S351000

Reexamination Certificate

active

06632425

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to processes for the purification of proteins. More specifically, methods for solubilizing and purifying proteins expressed in an insoluble form using low concentrations of chaotropic agents, such as guanidine salts, are provided. Also provided are methods for refolding proteins solubilized according to the present invention.
2. Related Art
The advent of recombinant DNA technology has brought about an entirely new generation of protein products. The ability to clone and express proteins in bacteria, yeast and mammalian cells has made it possible to produce therapeutics and industrially important proteins at economically feasible levels. However, the expression of high levels of recombinant proteins in
Escherichia coli
often results in the formation of inactive, denatured protein that accumulates in intracellular aggregates known as insoluble inclusion bodies. (Krueger et al., “Inclusion bodies from proteins produced at high levels in
Escherichia coli
,” in
Protein Folding
, L. M. Gierasch and P. King (Eds), Am. Ass. Adv. Sci., 136-142 (1990); Marston,
Biochem. J.
, 240:1-12 (1986); Mitraki, et al.,
Bio/Technol.
7:800-807 (1989); Schein,
Bio/Technol.
7:1141-1147 (1989); Taylor, et al.,
Bio/Technol.
4:553-557 (1986)). Inclusion bodies are dense aggregates, which are 2-3 &mgr;m in diameter and largely composed of recombinant protein, that can be separated from soluble bacterial proteins by low-speed centrifugation after cell lysis (Schoner, et al.
Biotechnology
3:151-154 (1985)).
The recovery of recombinantly expressed protein in the form of inclusion bodies has presented a number of problems. First, although the inclusion bodies contain a large percentage of the recombinantly produced protein, additional contaminating proteins must be removed in order to isolate the protein of interest Second, the proteins localized in inclusion bodies are in a form that is not biologically active, presumably due to incorrect folding.
Several methods have been developed to obtain active proteins from inclusion bodies. These strategies include the separation and purification of inclusion bodies from other cellular components, solubilization and reduction of the insoluble material, purification of solubilized proteins and ultimately renaturation of the proteins and generation of native disulfide bonds. The art teaches that concentrations of 6 M or greater of chaotropic agents, such as guanidine hydrochloride, guanidine isothiocyanate or urea are necessary for solubilization of the insoluble recombinant polypeptides from the inclusion bodies. See, for example, Vandenbroeck et al.,
Eur. J Biochem.
215:481-486 (1993); Meagher et al.,
Biotech. Bioeng.
43:969-977 (1994); Yang et al., U.S. Pat. No. 4,705,848, issued Nov. 10, 1987; Weir et al.,
Biochem. J.
245:85-91 (1987); and Fischer,
Biotech. Adv.
12:89-101 (1994).
Contrary to the teachings of the prior art, the inventors have discovered that low concentrations of guanidine salts can be used to solubilize biologically active (i.e., correctly folded) proteins and extract this population of the protein from a heterogeneous protein mixture localized in inclusion bodies. The methods disclosed herein are particularly useful for the purification of chemokines.
SUMMARY OF THE INVENTION
The present invention relates to the use of low concentrations of guanidine salts to solubilize inclusion bodies comprising target proteins. The purification method of the present invention results in a highly homogeneous product with no aggregated form of the target protein and can be easily scaled up and adapted for cGMP manufacturing. Further, the recovered product has a high purity, extremely low endotoxin levels as well as low levels of residual DNA.
According to the invention, inclusion bodies are released from cells by lysis, optionally washed to remove cellular components prior to extraction, and extracted with solutions containing low concentrations of guanidine salts.
More specifically, the invention provides for methods for solubilizing inclusion bodies by treatment with guanidine salts at concentrations of about 0.7 to about 3.5 M. The present invention also provides methods for solubilizing inclusion bodies by treatment with guanidine salts at concentrations of about 1 to about 2 M.
In another aspect, methods are provided for refolding target proteins which have been solubilized using guanidine salts. These methods involve the rapid dilution of guanidine salt extracts and optionally employ agents which facilitate target protein refolding. The invention further relates to the purification of solubilized target proteins using tandem column techniques.
The methods of the invention have the advantage of offering uniformity of equipment requirements for any desired product. While conditions required for optimum efficiency of solubilization and refolding will vary with each protein, the present invention is applicable, with only minor modification, for any protein which forms inclusion bodies. The modifications required to achieve optimum solubilization and refolding conditions for specific proteins are disclosed herein or are will be readily apparent to the skilled artisan after reading the present specification.


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