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
1999-08-16
2002-03-26
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...
C435S254200, C435S252300, C435S320100, C435S325000, C435S348000, C435S471000, C435S483000
Reexamination Certificate
active
06361969
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an expression system which provides heterologous proteins expressed by a non-native host organism but which have native-protein-like biological activity and/or structure.
BACKGROUND TO THE INVENTION
Advances during the past decade in molecular biology and genetic engineering have made it possible to produce large amounts of protein products using heterologous expression systems.
The use of heterologous hosts for production of, for example, therapeutic proteins, can lead, however, to differences in the biological and/or structural properties of the recombinant product. Amongst the biochemical modifications that commonly occur to proteins during or following their synthesis in the cell, the formation of disulphide bonds is of relevance since this modification is coupled to the correct folding or assembly of disulphide-bonded proteins (reviewed by J. C. A. Bardwell and J. Beckwith,
Cell
, 74: 769-771, 1993; R. B. Freedman, in
Protein Folding
, T. E. Creighton (ed.), W. H. Freeman and Co., New York, pp. 455-539, 1992).
In bacteria and other host cells for example, under certain conditions, some heterologous proteins are precipitated within cells as “retractile” or “inclusion” bodies. Such refractile or inclusion bodies consist of dense masses of partially folded, reduced heterologous protein which is often in a form which is not biologically active (S. B. Storrs et al.,
Protein Folding—American Chemical Society Symposium Series
470, Chapter 15: 197-204, 1991). It is believed that the biological inactivity of natively-disulphide-bonded refractile or inclusion heterologous proteins is due to incorrect protein folding or assembly brought about by the non-formation or misformation of the disulphide bonds within the proteins. The biological inactivity of refractile or inclusion heterologous proteins due to the process of incorrect protein folding or assembly is believed to occur either before or after intracellular precipitation or during isolation of the proteins.
Moreover, very often the biological function of a protein is regulated or at least influenced by the state of oxidation of its sulphydryl groups. This is the case for some enzymatic activities where the reversibility and timing of oxidation of sulphydryl groups has been proposed as a physiological control mechanism.
There are numerous examples of disulphide-bonded proteins in the literature. For instance, most viral glycoproteins and some growth factors are known to be disulphide-bonded. In general, disulphide bonds are essential to correct protein folding. Examples of disulphide-bonded recombinant heterologous proteins that have been shown to be misfolded when expressed in, for example, yeast cells include hepatitis B virus large surface protein (Biemans et al.,
DNA Cell Biol
., 10: 191-200, 1991), &agr;-1-antitrypsin (Moir and Dumais,
Gene
, 56: 209-217, 1987), and erythropoietin (Elliott et al.,
Gene
, 79: 167-180, 1989). Examples of recombinant proteins expressed in, for example, insect or mammalian cells, for which disulphide bonds have been shown to be essential for correct protein folding, include granulocyte/macrophage colony stimulating factor (GM-CSF) (Kaushansky et al.,
Proc. Natl. Acad. Sci. USA
, 86: 1213-1217, 1989), Friend erythroleukaemia virus (SFFV) glycoprotein gp55 (Gliniak et al.,
J. Biol. Chem
., 266: 22991-22997, 1991), glycoprotein of vesicular stomatitis virus (VSV-G) (Grigera et al.,
J. Virol
., 66: 3749-3757, 1992), pulmonary surfactant protein D (Crouch et al.,
J. Biol. Chem
., 269: 15808-15813, 1994), low density lipoprotein (LDL) receptor (Bieri et al.,
Biochemistry
, 34: 13059-13065, 1995), insulin-like growth factor (Nahri et al.,
Biochemistry
, 32: 5214-5221, 1993), and angiotensin-converting enzyme (ACE) (Sturrock et al.,
Biochemistry
, 35: 9560-9566, 1996). It should be noted that in all these cases, heterologous protein expression in particular host cells was only used to produce sufficient quantities of the protein concerned to enable structural studies to be carried out.
Several protein factors which catalyze disulphide bond formation have been characterized. Protein disulphide isomerase (PDI) is an abundant, multifunctional protein found in the lumen of the endoplasmic reticulum (ER) that promotes proper formation of disulphide bonds in secretory and cell surface proteins (LaMantia et al.,
Proc. Natl. Acad. Sci. USA
, 88: 4453-4457, 1991; Farquhar et al.,
Gene
, 108: 81-89, 1991; Freedman,
Cell
, 57: 1069-1072, 1989;
Laboissière et al.,
J. Biol. Chem
., 270: 28006-28009, 1995).
A similar function, but in a different cellular compartment, has been ascribed to another small, ubiquitous protein, thioredoxin (TRX) (Gan,
J. Biol. Chem
., 266: 1692-1696, 1991; Muller,
J. Biol. Chem
., 266: 9194-9202, 1991; Chivers et al.,
EMBO J
., 15: 2659-2667, 1996), that has an active-site sequence similar to that of PDI. Thioredoxins are cytosolic polypeptides capable of catalyzing the reduction of disulphides using glutathione as a reductant (Holmgren,
J. Biol. Chem
., 264: 13963-13966, 1989). It has been postulated that thioredoxin may also be involved in the reduction of prematurely formed disulphides in proteins that have entered the ER. Since the biological activity of a number of key enzymes involved in crucial metabolic pathways depends on the cytosolic redox system, it is plausible that TRX plays a relevant role in the modification of proteins involved in folding in cellular compartments other than the cytosol.
In the numerous organisms, for example, bacteria, yeast, mammalian cells and insect cells, which have been genetically manipulated to (over)express heterologous proteins, the problem encountered with most expression systems is the inability to express proteins which are biologically active.
SUMMARY OF THE INVENTION
It now appears that due to the lack of or inefficient amount of the enzymes necessary for correct folding or assembly of heterologous proteins in non-native expression hosts, such expressed heterologous proteins are often not biologically active and/or have an incorrect protein structure.
The present invention overcomes this problem and allows for the expression of biologically active and/or correctly structured heterologous proteins in non-native expression hosts.
It has now been found that expression cassettes encoding PDI or TRX can be used to transform a host organism thereby making it capable of overexpressing PDI or TRX. Preferably, the host organism is yeast. Yeast cells, for example, overexpressing these proteins can subsequently or simultaneously be transformed with expression vectors encoding one or more desirable heterologous proteins. The heterologous proteins expressed in such PDI/TRX-transformed yeast cells are in a properly-folded, biologically active form due to the disulphide bond formation activity of the PDI or TRX enzymes co-expressed in the same cell.
Such systems for producing biologically active heterologous proteins can be advantageously used for the production of, for example, proteins for human or veterinary therapeutic and/or diagnostic use or other proteins of commercial or research interest. The correct and optimum biological activity effected by the methods of the present invention is paramount in producing, for example, effective drugs and diagnostic reagents.
PDI overexpression in
Saccharomyces cerevisiae
has been found to enhance the secretion of human platelet-derived growth factor B homodimer (PDGF-BB) into the culture medium (Robinson et al.,
Bio/Technology
, 12: 381-384, 1994).
In the present invention an increased level of heterologous protein folding efficiency in cells has been demonstrated.
According to a first aspect of the invention there is provided a vector comprising an expression cassette comprising a DNA sequence encoding a protein capable of catalyzing disulphide bond formation.
Such a vector desirably results in (over)expression of the protein in a transformed host cell, thus providing the conditions for correct heterologous protein folding.
The pro
Blackburn Robert P.
Chiron S.p.A.
Harbin Alisa A.
Schwartzman Robert A.
Valla S. Maurice
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