Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-11-23
2001-03-27
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023100, C536S023400, C536S024100, C530S350000, C435S069100, C435S252300, C435S254110, C435S252330, C435S091400, C435S320100, C435S325000, C435S348000
Reexamination Certificate
active
06207420
ABSTRACT:
BACKGROUND
The present invention relates to recombinant methods of producing fusion proteins using
E. coli
proteins as carrier proteins for producing soluble fusion proteins.
A major benefit resulting from the advent of recombinant DNA technology has been the large scale production of proteins of medical or industrial importance. The simplest and most inexpensive means available for obtaining large amounts of proteins by recombinant DNA technology is by expression of protein genes in bacteria (Georgiou and Valax, 1996). The efficient synthesis of heterologous proteins in the bacterium
Escherichia coli
has now become routine. However, when high expression levels are achieved, recombinant proteins are frequently expressed in
E. coli
as insoluble protein aggregates termed “inclusion bodies.” Although initial purification of inclusion body material is relatively simple, the protein must be subsequently refolded into an active form, which is typically a cumbersome trial-and-error process (Georgiou and Valax, 1996). Thus, it is much more desirable to express the recombinant protein in soluble form.
A strategy to avoid inclusion body formation is to fuse the protein of interest (i.e. the target protein) to a protein known to be expressed at substantial levels in soluble form in
E. coli
(i.e. the carrier protein) . The most widely used carrier protein for the purpose of solubilization is thioredoxin from
E. coli
(LaVallie et al., 1993). A fusion protein system using thioredoxin for solubilization of target proteins is now being marketed by Invitrogen Corporation (Carlsbad, Calif.). However, despite being touted for its ability to solubilize proteins in a fusion protein, thioredoxin does not always lead to formation of a fusion protein which is soluble at the normal
E. coli
growth temperature of 37° C. LaVallie et al. (1993) used thioredoxin as a carrier protein to express 11 human and murine cytokines. Of the 11 proteins, only 4 were expressed in soluble form as thioredoxin fusions at 37° C. The non-soluble fusion proteins with thioredoxin could be expressed in soluble form by reducing the growth temperature for expression to as low as 15° C. Several problems with the use of thioredoxin fusions for protein solubilization for any protein are apparent. For example, having to reduce the expression temperature to as low as 15° C. may give unacceptable low rates of protein expression and slow growth rates. Also, due to the small size of thioredoxin (11.7 kilodaltons), fusions with larger proteins may not be soluble; that is, thioredoxin may not be large enough to compensate for the insolubility of a large protein.
Two other
E. coli
fusion protein systems are widely used: fusions with
E. coli
maltose-binding protein (Guan et al., 1988), which is 40 kilodaltons in size, and fusions with
Schistosoma japonicum
glutathione S-transferase (Smith and Johnson, 198.8), 26 kilodaltons in size. Both of these systems were developed with the objective of enabling an affinity purification of the fusion protein to be carried out. Both systems tend to give soluble fusion proteins but fail to do so approximately 25% of the time (New England Biolabs Tech Data Sheet, 1992; Smith and Johnson, 1988). Thus, thioredoxin fusions appear to be more soluble than either maltose-binding protein fusions or glutathione S-transferase fusions. An
E. coli
fusion protein system which could be reliably produced in a soluble form would be desirable.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a fusion sequence comprising a carrier protein comprising an
E. coli
protein having a predicted solubility probability of at least 90% fused to a target heterologous peptide or protein. The peptide or protein may be fused to the amino terminus of the soluble protein or the carboxyl terminus of the soluble protein. The fusion sequence according to this invention may optionally contain a linker peptide between the carrier protein and the selected peptide or protein. This linker provides, where needed, a selected cleavage site or a stretch of amino acids capable of preventing steric hindrance between the carrier protein and the target peptide or protein.
In another aspect, the present invention provides a DNA molecule encoding the fusion sequence defined above in association with, and under the control of, an expression control sequence capable of directing the expression of the fusion protein in a desired host cell, in particular,
E. coli.
Still a further aspect of the invention is a host cell (especially
E. coli
) transformed with, or having integrated into its genome, a DNA sequence comprising a DNA encoding a carrier protein as defined herein fused to the DNA sequence of a selected heterologous peptide or protein. This fusion sequence is desirably under the control of an expression control sequence capable of directing the expression of a fusion protein in the cell.
In yet another aspect, there is provided herein a novel method for increasing the expression of soluble recombinant proteins. The method includes culturing under suitable conditions the above-described host cell to produce the fusion protein.
In one embodiment of the method contemplated herein, if the resulting fusion protein is cytoplasmic, the cell can be lysed by conventional means to obtain the soluble fusion protein. More preferably in the case of cytoplasmic fusion proteins, the method includes releasing the fusion protein from the host cell by a method such as sonication or homogenation. The fusion protein is the purified by conventional means. In still another embodiment, if a secretory leader is employed in the fusion protein construct, the fusion protein can be recovered from a periplasmic extract or from the cell culture medium (using osmotic shock to release the protein). As yet a further step in the above methods, the desired heterologous protein can be cleaved from fusion with the carrier protein by conventional means.
Other aspects and advantages of the present invention will be apparent upon consideration of the following detailed description of preferred embodiments.
In particular, the objective of the present invention is to improve the purification process of recombinant fusion proteins by avoiding the initial expression of these fusion proteins in
E. coli
as insoluble inclusion bodies by using
E. coli
carrier proteins having predicted solubility probabilities of at least 90%.
REFERENCES:
patent: 5270181 (1993-12-01), McCoy et al.
patent: 5292646 (1994-03-01), McCoy et al.
patent: 9402502 (1994-02-01), None
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David L. Wilkinson and Roger G. Harrison, “Predicting the Solubility of Recombinant Proteins inEscherichia Coli”,Biotechnology, 9:443-448, May 1991.
Donahue et al., “Human IL-3 and GM-CSF Act Synergistically in Stimulating Hematopoiesis in Primates”,Science, 241:1820-1823, Sep. 1988.
Lutsenko et al., “Recombinant Interleukin-3 Expression System inE. coli”, http://www.ncbi.nlm.nih.gov/htbin-post/Entrez/query?uid=1524590&form=6&db=m&Dopt=r,Bioorg Khim, 18 (3) :391-397, Mar. 18, 1992 (page one only).
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Joost Haelewyn and Marc De Ley, “A Rapid Single-Step Purification Method for Human Interferon-&ggr; from IsolatedEscherichia coliInclusion Bodies”,Biochemistry and Molecular Biology International, 37 (6) :1163-1171, Dec. 1995.
Sharma et al., “Folding and Activation of Recombinant Human Prorenin”,Biotechnology and Applied Biochemistry, 9:181-193, 1987.
Guan et al., “Vectors That Facilitate the Expression and Purification of Foreign Peptides inEscherichia coliby Fusion to Maltose-Binding Protein”,Gene, 67:21-30, 1988.
Zhang et al., “Expression and Functionqal Characterization ofEscherichia coliNusQ and Lambda Q as Glutathio
Davis Gregory D.
Harrison Roger G.
Dunlap, Codding and Rogers P.C.
Gansheroff Lisa
Schwartzman Robert A.
The Board of Regents of the University of Oklahoma
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