Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1993-07-27
2001-12-25
Yucel, Remy (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S069100, C435S071100, C435S471000
Reexamination Certificate
active
06333175
ABSTRACT:
DESCRIPTION
The present invention concerns a process for increasing the formation of the natural protein conformation when disulfide-bonded proteins are secreted by a prokaryotic host organism that contains a recombinant DNA coding for the secreted protein.
Protein synthesis or translation in prokaryotic microorganisms occurs at the ribosomes in the cytoplasm. When recombinant DNA is expressed in bacterial host organisms it is often desirable that the resulting recombinant gene product or protein is secreted from the cytoplasm through the inner bacterial membrane into the periplasmatic space between the inner and outer membrane. The secreted proteins can then for example be released from the periplasm into the culture medium by an osmotic shock. A disadvantage of this process is that when disulfide-bonded proteins are secreted the native or natural conformation is not usually formed correctly i.e. polypeptides are produced with a false or incomplete formation of the disulphide bridges which are biologically inactive.
Thiol reagents are used in methods for the in vitro renaturation of insoluble proteins which form as inclusion bodies in the cytoplasm when recombinant DNA is expressed in prokaryotic cells. It is known that the thiol reagents are rapidly oxidized to disulphides in the presence of atmospheric oxygen.
An object of the present invention was to increase the formation of the natural protein conformation when disulfide-bonded proteins are secreted.
In particular promoting the formation of the native protein conformation could replace the in vitro renaturation of falsely disulfide-bonded proteins which can be very complicated.
The object according to the present invention is achieved by a process for increasing the formation of the natural protein conformation when disulfide-bonded proteins are secreted by a prokaryotic host organism that contains a recombinant DNA coding for the secreted protein, wherein the host organism is cultured in a suitable culture medium under conditions suitable for the expression of the recombinant DNA whereby the process is characterized in that a culture medium containing 0.1 to 20 mmol/l of one or several thiol reagents is used.
Surprisingly it is possible by using the process according to the present invention to increase the yield of correctly disulfide-bonded proteins by adding thiol reagents to the fermentation medium. The process according to the present invention can be used for all proteins which contain one or several disulphide bridges. In particular when only a small amount of protein is required, e.g. in the production of growth factors on a mg scale (nerve growth factor, interleukins or similar compounds), an in vitro renaturation can be dispensed with by using the process according to the present invention.
A culture medium containing 0.1 to 20 mmol/l of one or several thiol reagents has proven to be suitable for the process according to the present invention. If a culture medium is used which contains less than 0.1 mmol/l of thiol reagents then no appreciable increase in the formation of the natural protein conformation is found. The upper limit for the thiol concentration is about 20 mmol/l and is manifested by a substantial decrease in the yields of secreted functional protein as well as in a marked decline in cell growth. The culture medium used preferably contains 1 to 15 mmol/l and particularly preferably contains 3 to 12 mmol/l of one or several thiol reagents. If glutathione is used as the thiol reagent then about 5 mmol/l has proven to be an optimal concentration.
The term “thiol reagent” used in the present description means either a reducing thiol reagent with SH groups or a mixture of reducing thiol reagents with SH groups and oxidizing thiol reagents with disulfide groups.
Those which have a single SH group per molecule have proven to be particularly suitable as reducing thiol reagents. Particularly preferred substances are reduced glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, &bgr;-mercaptoethanol and similar compounds. Most preferred are N-acetylcysteine and reduced glutathione (GSH). The thiol reagents can either be used singly or in mixtures.
Although the sole use of reducing thiol reagents is generally preferred, the use of a mixture of reducing and oxidizing thiol reagents leads to an improved yield of correctly disulfide-bonded proteins. When such a mixture of reducing and oxidizing thiol reagents is used the molar ratio of reducing to oxidizing thiol reagents is preferably 2:1 to 20:1, particularly preferably 5:1 to 10:1. A mixture of a reduced and of an oxidized thiol reagent is for example reduced glutathione (GSH) and glutathione disulfide (GSSG).
An example of the process according to the present invention is the heterologous expression of the bifunctional &agr;-amylase/trypsin inhibitor from Eleusine coracana Gaertneri (RBI) in
E. coli
. The inhibitor was characterized by Shivaraj and Pattabiraman (Shivaraj B. & Pattabiraman, T. N., Indian J.Biochem.Biophys. 17 (1980), 181-193; Shivaraj B. & Pattabiraman, T. N., Biochem.J. 193 (1981), 29-36). The amino acid sequence of the inhibitor was disclosed by Campos and Richardson (Campos, F. A. P. & Richardson, M., FEBS Letters 152 (1983), 300-304). This protein is comprised of 122 amino acids, it contains 5 intramolecular disulfide bridges and belongs to a new a-amylase/trypsin inhibitor class (Halford et al., Biochim. Biophys. Acta 950 (1988), 435-440), whereby it is its only bifunctional member. At this point it should, however, be noted that the process according to the present invention can also be applied to the isolation of other secreted recombinant disulfide-bonded proteins (e.g. antibody fragments) in prokaryotic host organisms.
In order to isolate the secretory RBI protein in a functional form in
E. coli
a synthetic RBI gene was fused using genetic engineering methods to the signal sequence of the outer membrane protein A (OmpA) of
E. coli
and the fusion was expressed in
E. coli
on a recombinant plasmid under the control of a lac promoter. In this way the polypeptide chain of the recombinant protein is transported into the periplasm of the prokaryotic host cell where, after cleavage of the signal sequence, it can as a result of the oxidizing properties of this cell compartment fold to form the native protein with formation of the intramolecular disulfide bridges. However, only small amounts of the functional protein can be obtained by this folding. When thiol reagents are present in the culture medium according to the present invention it is, however, possible to considerably increase the yield of functional protein (by a factor of 5).
The host organism for the process according to the present invention is a prokaryotic host organism. The expression of the recombinant protein is preferably carried out in a gram-negative prokaryotic host organism, particularly preferably in
E. coli.
In the process according to the present invention it is in general preferable that the recombinant DNA coding for the secreted protein has an operative linkage with a DNA fragment that codes for a signal peptide for penetrating inner bacterial membranes. The term “operative linkage” within the sense of the present invention means that there is a translational fusion between the heterologous protein and the signal peptide. In this process the signal peptide usually forms the N-terminal part of the translational fusion. The type of signal peptide is not critical for the present invention apart from the fact that it should enable the secretion of the recombinant protein. A large number of such signal peptides are known to one skilled in the area of molecular biology. A series of signal peptides are enumerated for example by Winnacker (Gene und Klone, Eine Einfuhrung in die Gentechnologie, published by Chemie Verlag Weinheim (1985), p. 256). If the process according to the present invention is carried out in
E. coli
as the host organism then the signal peptide from the
E. coli
OmpA protein has proven to be particularly suitable.
For expression in a
Glockshuber Rudolf
Rudolph Rainer
Skerra Arne
Wunderlich Martina
Arent Fox Kintner & Plotkin & Kahn, PLLC
Roche Diagnostics GmbH
Yucel Remy
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