Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-07-24
2001-05-15
Romeo, David (Department: 1646)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024200, C536S024100, C536S023400, C435S320100, C435S252330, C435S254100, C435S254200, C435S069100, C435S069700, C530S353000, C514S002600, C514S012200
Reexamination Certificate
active
06232458
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the production of recombinant tropoelastins, and variants of these recombinant tropoelastins, from synthetic polynucleotides, and uses of the tropoelastins and variants.
BACKGROUND ART
There are various forms of tropoelastin that typically appear to consist of two types of alternating domains: those rich in hydrophobic amino acids (responsible for the elastic properties) and those rich in lysin residues (responsible for cross-link formation). Hydrophobic and cross-linking domains are encoded in separate exons (Indik et al. 1987).
The gene for tropoelastin is believed to be present as a single copy in the mammalian genome, and is expressed in the form of multiple transcripts, distinguished by alternative splicing of the pre-mRNA (Indik et al, 1990; Oliver et al, 1987).
Previous recombinant work with tropoelastin has been reported by Indik et al (1990) who achieved modest expression of a natural human tropoelastin sequence from cDNA. Their product was unstable, the free polypeptide being rapidly degraded.
Bressan et al (1987) have reported the cloning of a defined naturally occurring segment of chick tropoelastin.
DESCRIPTION OF THE INVENTION
The present invention provides for the expression of significant amounts of tropoelastins or variants of the tropoelastins in recombinant expression systems.
The present inventors have recognised that tropoelastins are proteins which can be used in a variety of, for instance, pharmaceutical applications, but these uses require significant quantities of tropoelastin. These quantities could be obtained by cloning naturally occurring tropoelastin genes, but the present inventors show how they can be more easily obtained by producing synthetic polynucleotides adapted to provide enhanced expression.
The present inventors have recognised that because tropoelastins have highly repetitive coding sequences, the tropoelastin genes have the potential to include significant numbers of codons which have low usage in particular hosts. Codons of low usage can hamper gene expression.
For example, in one tropoelastin coding sequence described in detail in this application, the natural sequence contains of the order of 80 glycine GGA codons which comprises 10% of the gene and have low usage in
Escherichia coli
[Fazio et al., 1988, and Genetics Computer Group (GCG) package version 7-UNIX using Codon Frequency and Gen Run Data: ecohigh-cod].
According to a first aspect of the present invention, there is provided a synthetic polynucleotide encoding the amino acid sequence of a tropoelastin or a variant of the tropoelastin.
The tropoelastin may be a mammalian or avian tropoelastin such as human, bovine, ovine, porcine, rat or chick tropoelastin. Preferably, the tropoelastin is human tropoelastin.
The synthetic polynucleotide sequence is altered with respect to the natural coding sequence for the tropoelastin molecule or variant so that:
a) it codes for a tropoelastin sequence or a variant of the tropoelastin; and
b) all or some of the codons which hamper expression in the expression system in which the polynucleotide is to be expressed, are replaced with codons more favourable for expression in the expression system.
Preferably all, or part, of the 5′ or 3′ untranslated regions, or both, of the natural coding sequence are excluded from the synthetic polynucleotide.
Preferably all, or part, of the signal peptide encoding region is excluded from the synthetic polynucleotide.
Where the synthetic polynucleotide is prepared from assembled oligonucleotides it is preferred to incorporate restriction sites in the sequence to facilitate assembly of the polynucleotide.
Restriction sites incorporated in the polynucleotide sequence are also useful for:
1. facilitating subcloning of manageable blocks for sequence confirmation;
2. providing sites for later introduction of modifications to the polynucleotide as insertions, deletions or base changes;
3. facilitating confirmation of correct polynucleotide assembly by restriction endonuclease digestion.
A preferred expression system is an
Escherichia coli
expression system. However, the invention includes within its scope synthetic polynucleotides suitable for use in other expression systems such as other microbial expression systems. These other expression systems include yeast and bacterial expression systems, insect cell expression systems, and expression systems involving other eukaryotic cell lines or whole organisms.
Modifications to codon usage to provide enhanced expression are discussed in:
Zhang et al (1991) for
E. coli
, yeast, fruit fly and primates where codon usage tables are provided;
Newgard et al (1986) for mammals; and Murray et al (1989) for plants. Preferred codon usages are indicated in these publications.
Preferably, at least 50% of codons for any particular amino acid are selected and altered to reflect preferred codon usage in the host of choice.
Preferably, the polynucleotide is a fused polynucleotide with the tropoelastin or variant encoding sequence fused to a polynucleotide sequence compatible with the host. The compatible sequence is preferably at the 5′ end of the polynucleotide molecule.
Preferred compatible polynucleotides include those which encode all or part of a polypeptide which causes the expressed fusion to be secreted or expressed as a cell surface protein so as to facilitate purification of the expressed product, or expressed as a cytoplasmic protein.
One preferred compatible polynucleotide is one encoding all or part of glutathione-S-transferase.
In addition the synthetic polynucleotides can encode additional residues such as an N-terminal methionine or f-methionine not present in the natural counterpart.
A preferred synthetic polynucleotide is one comprising the sequence illustrated in
FIGS. 3
(
1
) to
3
(
5
) (SEQ ID NO 1) or a part of it, encoding a polypeptide which retains elastic properties. The sequence illustrated in
FIGS. 3
(
1
) to
3
(
5
) is 2210 bp in size.
To our knowledge, this is the largest synthetic gene constructed so fat. Previously, the largest was of the order of 1.5 kb in size.
The actual changes made in this sequence in comparison with the natural sequence from which it was derived are shown in
FIGS. 6
(
1
) to
6
(
4
) comparing the synthetic sequence (SEQ ID NO 1) with the natural sequence (SEQ ID NO 53). Synthetic polynucleotides in which only some of the base changes shown in that Figure have been made are also within the scope of the invention.
It is known that tropoelastin genes in nature are expressed as multiple transcripts which are distinguished by alternative splicing of the pre-mRNA as described in, for instance:
Indik et al, 1990; Oliver et al, 1987; Heim et al, 1991; Raju et al, 1987; and Yeh et al, 1987. The tropoelastins of the present invention for which synthetic polynucleotides are prepared are intended to encompass these different splice forms.
Variants of tropoelastins embodying the present invention are polypeptides which retain the basic structural attributes, namely the elastic properties, of a tropoelastin molecule, and which are homologous to naturally occurring tropoelastin molecules. For the purposes of this description, “homology” between two sequences connotes a likeness short of identity indicative of a derivation of one sequence from the other. In particular, a polypeptide is homologous to a tropoelastin molecule if a comparison of amino-acid sequences between the molecules reveals an identity of greater than about 65% over any contiguous 20 amino acid stretch or over any repetitive element of the tropoelastin molecule shorter than 20 amino acids in length. Such a sequence comparison can be performed via known algorithms, such as the one described by Lipman and Pearson, Science 227: 1435 (1985) which are readily implemented by computer.
Variants of tropoelastins can be produced by conventional site-directed or random mutagenesis. This is one avenue for routinely identifying residues of the molecule that can be modified without destroying the
Martin Stephen Lewis
Weiss Anthony Steven
Howson and Howson
Romeo David
The University of Sydney
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