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
1995-05-17
2003-11-04
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069800, C435S069900, C435S320100, C435S325000, C435S254100, C435S254110, C435S254210, C530S324000, C530S350000, C530S399000, C536S023400, C536S023500
Reexamination Certificate
active
06642029
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
It is suspected that the somatic growth which follows the administration of growth hormone in vivo is mediated through a family of mitogenic, insulin-like peptides whose serum concentrations are growth hormone dependent. These polypeptides include somatomedin-C, somatomedin-A, and insulin-like growth factors I and II (IGF I and IGF II). IGF I and II can be isolated from human serum and have amino acid sequences which are broadly homologous to that of insulin. At present, only limited quantities of these growth factors may be obtained by separation from human serum. It would thus be of great scientific and clinical interest to be able to produce relatively large quantities of the growth factors by recombinant DNA techniques.
2. Description of the Prior Art
The amino acid sequences for human insulin-like growth factors I and II (IGF I and II) were first determined by Rinderknecht and Humbel (1978) J. Biol. Chem. 253:2769-2776 and Rinderknecht and Humbel (1978) FEBS Letters 89:283-286, respectively. The nature of the IGF receptors is discussed in Massague and Czech (1982) J. Biol. Chem. 257:5038-5045. Kurjan and Herskowitz, Cell (1982) 30:933-934 describe a putative &agr;-factor precursor containing four tandem copies of mature &agr;-factor, describing the sequence and postulating a processing mechanism. Kurjan and Herskowitz, Abstracts of Papers presented at the 1981 Cold Spring Harbor Meeting on The Molecular Biology of Yeast, p. 242, in an Abstract entitled, “A Putative Alpha-Factor Precursor Containing Four Tandem Repeats of Mature Alpha-Factor,” describe the sequence encoding for the &agr;-factor and spacers between two of such sequences.
SUMMARY OF THE INVENTION
Methods and compositions are provided for the efficient production of mature human insulin-like growth factor (IGF). In particular, expression of a “pre”-IGF I and “pre”-IGF II in a yeast host facilitates secretion of the polypeptides into the nutrient medium. DNA constructs are generated by joining DNA sequences from diverse sources, including both natural and synthetic sources. The resulting DNA constructs are stably replicated in the yeast and provide efficient, high level production of processed “pre”-polypeptides which may be isolated in high yield from the nutrient medium.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
DNA sequences capable of expressing human insulin-like growth factors (IGF I and II) are provided. These DNA sequences can be incorporated into vectors, and the resulting plasmids used to transform susceptible hosts. Transformation of a susceptible host with such recombinant plasmids results in expression of the insulin-like growth factor gene and production of the polypeptide product.
In particular, novel DNA constructs are provided for the production of the precursor polypeptides (“pre”-IGF I and “pre”-IGF II) in a yeast host capable of processing said precursor polypeptides and secreting the mature polypeptide product into the nutrient medium. The DNA constructs include a replication system capable of stable maintenance in a yeast host, an efficient promoter, a structural gene including leader and processing signals in reading frame with said structural gene, and a transcriptional terminator sequence downstream from the structural gene. Optionally, other sequences can be provided for transcriptional regulation, amplification of the gene, exogenous regulation of transcription, and the like. By “pre”-IGF I and “pre”-IGF II, it is meant that the DNA sequence encoding for the mature polypeptide is joined to and in reading frame with a leader sequence including processing signals efficiently recognized by the yeast host. Thus, “pre” denotes the inclusion of secretion and processing signals associated with a yeast host and not any processing signals associated with the gene encoding the polypeptide of interest.
In preparing the DNA construct, it is necessary to bring the individual sequences embodying the replication system, promoter, structural gene including leader and processing signals, and terminator together in a predetermined order to assure that they are able to properly function in the resulting plasmid. As described hereinafter, adaptor molecules may be employed to assure the proper orientation and order of the sequences.
The IGF I and IGF II genes which are employed may be chromosomal DNA, cDNA, synthetic DNA, or combinations thereof. The leader and processing signals will normally be derived from naturally occurring DNA sequences in yeast which provide for secretion of a polypeptide. Such polypeptides which are naturally secreted by yeast include &agr;-factor, a-factor, acid phosphatase and the like. The remaining sequences which comprise the construct including the replication system, promoter, and terminator, are well known and described in the literature.
Since the various DNA sequences which are joined to form the DNA construct of the present invention will be derived from diverse sources, it will be convenient to join the sequences by means of connecting or adaptor molecules. In particular, adaptors can be advantageously employed to connect the 3′-end of the coding strand of the leader and processing signal sequence to the 5′-end of the IGF coding strand together with their respective complementary DNA strands. The leader and processing signal sequence may be internally restricted near its 3′-terminus so that it lacks a predetermined number of base pairs of the coding region. An adaptor can then be constructed so that, when joining the leader and processing sequence to the IGF coding strand, the missing base pairs are provided and the IGF coding strand is in the proper reading frame relative to the leader sequence. The synthetic IGF coding region and/or the adaptor at its 3′-end will provide translational stop codons to assure that the C-terminus of the polypeptide is the same as the naturally occurring C-terminus.
The adaptors will have from about 5 to 40 bases, more usually from about 8 to 35 bases, in the coding sequence and may have either cohesive or blunt ends, with cohesive ends being preferred. Desirably, the termini of the adaptor will have cohesive ends associated with different restriction enzymes so that the adaptor will selectively link two different DNA sequences having the appropriate complementary cohesive end.
The subject invention will be illustrated with synthetic fragments coding for IGF I and IGF II joined to the leader and processing signals of yeast &agr;-factor. The yeast &agr;-factor may be restricted with HindIII and SalI. HindIII cleaves in the processing signal of the &agr;-factor precursor, cleaving 3′ to the second base in the coding strand of the glu codon, while the HindIII recognition sequence completes the glu codon, encodes for ala and provides the first 5′ base of the amino-terminal trp codon of mature &agr;-factor. With reference to the direction of transcription of the &agr;-factor gene, the SalI site is located upstream of the transcriptional terminator.
The synthetic genes coding for IGF will have nucleotide sequences based on the known amino acid sequences of the IGF I and IGF II polypeptides. Preferably, the synthetic sequences will employ codons which are preferentially utilized by the yeast host, e.g., based on the frequency with which the codons are found in the genes coding for the yeast glycolytic enzymes. Conveniently, the synthetic sequence will include cohesive ends rather than blunt ends for insertion into a restriction site in a cloning vehicle. Furthermore, restriction sites will be designed into the synthetic sequences using silent mutations in order to generate fragments which may be annealed into sequences capable of producing IGF I/IGF II hybrid peptide molecules.
In the examples, the synthetic fragments are provided with cohesive ends for EcoRI and inserted into the EcoRI site in pBR328. Usually, the synthetic sequence will include additional restriction sites proximal to each end of the polypeptide coding region. Such interior restriction si
Mullenbach Guy
Valenzuela Pablo D. T.
Allen Marianne P.
Blackburn Robert P.
Chiron Corporation
Guth Joseph H.
Robins Roberta L.
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