Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – Nonplant protein is expressed from the polynucleotide
Reexamination Certificate
1998-06-09
2002-02-05
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
Nonplant protein is expressed from the polynucleotide
C800S278000, C800S298000, C435S410000, C435S419000, C435S430000, C435S468000, C536S023100, C536S023500
Reexamination Certificate
active
06344600
ABSTRACT:
FIGS. 7 and 8
of the patent or application are photographs. Copies of this patent or patent application publication with photographs will be provided by the Office upon request and payment of the necessary fee.
BACKGROUND OF THE INVENTION
The invention relates to a method for producing hemin proteins using plant cells, and in particular the hemin proteins capable of reversibly binding oxygen, for example hemoglobin and its derivatives, and myoglobin. It relates, in addition, to the proteins obtained using the method. The invention also relates to the genetically transformed cells and plants capable of producing these proteins, and to the nucleic acid constructs involved in the transformation. In addition, the invention relates to products, for example pharmaceutical or cosmetic products, containing these hemin proteins.
BACKGROUND OF THE INVENTION
Hemin proteins are complex molecules composed of one or more polypeptide chain(s) in association with one or more ferroporphyrin nucleus or nuclei. These nuclei are composed of four pyrrole rings, juxtaposed in a closed structure and linked by methene bridges, and containing an iron atom at the center of the molecule. Hemin proteins differ from one another in the nature and the number of the polypeptide chains and in the nature of the side chains carried by the eight &bgr; atoms of the pyrrole rings. An example of a ferroporphyrin nucleus is iron-containing protoporphyrin IX, also known by the name “protoheme” or simply “heme” (FIG.
1
).
The hemin protein family comprises numerous substances which are important from the biological point of view in animals and in plants, particularly hemoglobin, myoglobin, cytochromes, peroxidases and catalases.
Hemoglobin is the main constituent of the red blood cells. Its essential function is to bind, transport and deliver the quantity of oxygen necessary for normal tissue function.
The hemoglobin molecule is composed of two types of globin chains or subunits, called &agr; and &bgr; (of 141 and 146 amino acids respectively), and linked in pairs to form a &agr;
2
&bgr;
2
tetramer. Each of these subunits contains, solidly attached in a hydrophobic sac, a heme molecule (that is to say protoporphyrin IX) containing, at the center, a divalant iron atom (Fe
2+
) to which a molecule of oxygen reversibly binds. Each tetrameric hemoglobin molecule therefore contains 4 iron atoms and 4 oxygen molecules which it binds during its passage through the lungs. The molecular mass of the tetramer is 64,650 D. In man, the &agr; and &bgr; chains are synthesized from two types of genes situated on chromosomes 16 and 11 respectively.
The term beta, or “nonalpha”, type chains covers not only the beta chains, but also the chains called epsilon, gamma or delta.
Normally, in adults, more than 95% of the hemoglobin consists of alpha
2
beta
2
tetramer, that is to say the association of two heterologous alpha-beta dimers, associated with the catalytic complex, heme. 2% to 3% of a hemoglobin consisting of alpha
2
delta
2
tetramers, and traces of fetal hemoglobin alpha
2
gamma
2
exist.
The tetrameric human hemoglobin molecule exists in two quaternary forms or structures depending on whether oxygen is bound or not to the iron atoms. In the presence of oxygen, hemoglobin is said to be in an R (for relaxed) state and its affinity for oxygen is high. In the absence of oxygen, hemoglobin is said to be in a T (for tense) state and its affinity for oxygen is 100 times lower (Perutz, 1970). The resultant affinity is linked to the equilibrium between the concentrations of R and T forms. The higher the concentration of hemoglobin in the T form at any level of oxygenation, the lower this affinity. The affinity of hemoglobin for oxygen is regulated by the cofactor-2,3-diphosphoglycerate (DPG), a small molecule derived from the metabolism of glucose and which binds to the &bgr; chains of tetrameric hemoglobin, reducing its affinity for oxygen.
The increase in the risks of infection by products derived from human blood (hepatitis, HIV) makes the development of an artificial oxygen carrier as substitute for blood transfusion necessary.
Techniques using recombinant DNAs have been proposed for producing the protein chains of globin.
The aim of the first techniques developed was essentially to cause the alpha and beta chains to be synthesized in
E. coli
separately (Naga{umlaut over (i )}and Thogen-Sen, 1987), involving cumbersome methods for separate expression of each of the chains. These methods could hardly be exploited on an industrial scale.
More recently, the expression of soluble and functional recombinant hemoglobin has been developed in
E. coli
(Hoffman et al., 1990, P.N.A.S., 87, 8521-8525) and
Saccharomyces cerevisiae
(Wagenbach et al., 1991, Biotechnology, 9, 57-61). Each of these systems has advantages and disadvantages. Indeed, the highest expression levels are obtained in
E. coli
which has, nevertheless, the disadvantage of producing endotoxins and of not cleaving the NH
2
terminal methionines contrary to
Saccharomyces cerevisiae
. In the yeast, the yields of synthesis of hemoglobin are low (3 to 5%), compared with the 10-15% obtained in
E. coli
. This currently limits the use of yeast in the context of an industrial development plan.
The use of animal cells in culture or of transgenic animals as hosts for production has also been achieved (Swanson et al., Bio/Technology, May 1992, 10, page 55). It appears that these techniques cannot currently be exploited because of low expression levels and the risks of contaminations by viruses and by prions.
The technical problem which the present invention proposes to solve is to produce hemin proteins, and in particular hemoglobin and its derivatives, in a large quantity at low costs, without the risk of viral or subviral contaminations. The inventors have provided a solution to this problem by using plant cells as host for the transformation and the production.
Various teams have already taken an interest in the production of mammalian recombinant proteins in plant cells or in transgenic plants. For example, the specific expression, in rapeseed, of leuenkephalin has been obtained with expression levels of about 0.1% (Vanderkerckhove et al., Biotechnology, 1989, 7, 929-932).
In 1990, Sijmons et al., (Biotechnology, 1990, 8, 217-221) transferred the gene for human serum albumin into tobacco and potato cells. Regardless of the origin of the signal peptides (human or plant), human serum albumin levels of the order of 0.02% of the total proteins were obtained in the potato leaves, stems and tubers.
Other mammalian recombinant proteins have also been produced in plants: hepatitis B surface antigen (Mason et al., P.N.A.S., 1992, 89, 11745-11749); human interferon (Edelbaum J. of Interferon Res., 1992, 12, 449-453); a mouse antibody to
Streptococcus mutans
, an agent for dental caries (Hiatt and Mass., FEBS, 1992, 307, 71-75); an anti-Herpes antibody (Russel, 1994) and hirudin (Moloney, 1994).
All these research studies show that the production of mammalian recombinant proteins in plant cells is possible and that the mechanisms of protein synthesis from the DNA sequences are similar in animal cells and plant cells.
On the other hand, little information is available on the subject of the iron-containing porphyrins in plants, particularly on their structures, their synthesis pathways and the assembly of the porphyrin nuclei and the protein chains to form the hemin proteins. The production of recombinant molecules having the capacity to reversibly bind oxygen, and requiring the assembly, in the cell, of heterologous proteins and of endogenous plant porphyrins has never been described.
SUMMARY OF THE INVENTION
The invention relates to a method for producing recombinant hemin proteins using plant cells. According to the method of the invention, the plant cell is genetically modified so as to be able to express the protein component of a hemin protein. The porphyrin nucleus is produced by the cell endogenously, the assembling of the protein and porphyrin components taking place
Baudino Sylvie
Dieryck Wilfrid
Gruber Veronique
Lenee Philippe
Marden Michael
LandOfFree
Method for producing human hemoglobin proteins using plant... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for producing human hemoglobin proteins using plant..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for producing human hemoglobin proteins using plant... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2974406