Production of biologically active recombinant insulin-like...

Details Production of biologically active recombinant insulin-like... Production of biologically active recombinant insulin-like...

1999-08-26

2001-05-22

Saoud, Christine J. (Department: 1647)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Insulin; related peptides

C435S069400, C435S320100, C930S010000

Reexamination Certificate

active

06235874

ABSTRACT:

FIELD OF THE INVENTION
The present invention (published in
DNA and Cell Biology
(1997), 16:883-892, hereinafter incorporated by reference) relates to a fish insulin-like growth factor II (IGF-II) cDNA and the biologically active IGF-II polypeptide expressed by said cDNA, and more particularly, relates to a recombinant tilapia IGF-II cDNA and the expression of said recombinant IGF-II cDNAs in cells. The IGF-II, signal, and E domain peptides can be produced by gene expression in
E. coli
, yeast, baculovirus, and fish cells or by in vitro synthesis. The E domain peptide has demonstrated mitogenic and anti-tumor effects.
BACKGROUND OF THE INVENTION
Insulin-like growth factors (IGFs) are mitogenic peptide hormones which play important roles in the growth and differentiation of vertebrates. IGFs are translated as a prepropeptide, which can be divided into an N-terminal prepeptide (which is also called a signal or leader peptide), a B to D domain polypeptide (which contains peptides from B, C, A and D domains), and a C-terminal E-domain peptide (which is also called a trailer peptide). The IGF signal and E peptides are proteolytically removed from the B to D polypeptide during protein maturation (Shamblott and Chen (1992),
Proc. Natl. Acad. Sci
. USA, 89:8913-8917). For this reason, the B to D domain polypeptide is also called “the IGF mature peptide”.
As of this time, there has been no reports concerning the functions of the IGF signal and E peptides. However, it is well known in the art that a signal peptide may facilitate the transport of protein out of the cell membrane. As for the E peptide, since the E peptide is a part of the IGF preproprotein and may actually be secreted as a part of an intact prohormone, it is possible that the E peptide may affect the processing, transport, or secretion of the mature peptide. It is also possible that the E peptide may indirectly or directly modulate the degradation, receptor interaction, or binding protein interactions of the mature peptide.
There are two kinds of IGFs, namely, IGF-I and IGF-II. IGF-I and IGF-II share high homology of protein folding structure and similar growth promotion effects, even though they are mediated by different IGF receptors (i.e., IGF-I is mediated by a tyrosine kinase receptor, whereas IGF-II is mediated by a mannose-6-phosphate receptor). IGF-I is a 70 amino acid polypeptide which mediates the growth-promoting actions of growth hormone as well as having important local paracrine and autocrine roles in multiple organs (Kavsan et al. (1993),
DNA and Cell Biology,
12:729-737).
In mammals, the mature form of IGF-II is a neutral protein consisting of 67 amino acid residues with three disulfide bonds. IGF-II is a single-chain polypeptide that contains a NH
2
—B—C—A—D—COOH domain. The signal peptide and E domain are removed from the mature IGF-II peptide. The cDNA sequences of IGF-II have been reported highly conserved in different mammals, including chickens, sheep, mice, humans, and rats. The mammalian IGF-II is primarily produced in liver during prenatal development under the control of placental lactogen (Gray et al. (1987),
DNA,
6:283-295). Because of its role as a key component regulating fetal growth, IGF-II is also called a “fetal growth factor”.
IGF-II has a complex gene structure. In humans, IGF-II gene consists of 10 exons and spans about 30 kb of DNA. The DNA sequence encoding the mature IGF-II polypeptides in humans is contained within exons 8, 9, and 10. In rat and mouse, IGF-II genes consist of 6 exons and span about 12 kb of DNA and the DNA sequence encoding the prepropeptide of rat or mouse IGF-II is contained within exons 4, 5, and 6.
Up until now, the gene structure and protein function of IGF-II in fish have remained unidentified. IGF-II like peptides have been reported present in the insulin cells of the elasmobranchian endocrine pancreas of fish (Reinneke et al. (1994),
Histochemistry
, 102:365-371). However, whether IGF-II is functioned as autocrine or paracrine is so far unclear. In addition, although so far two IGF-II cDNAs have been discovered in the liver of two fish species:
Sparus aurata
(Duguay et al. (1996),
J. Mol. Endocrinol
., 16:123-132) and rainbow trout (Shamblott and Chen (1993), supra), there has been no report or study relating to the expression of fish IGF-II in cells as well as the physiological activity of fish IGF-II in vivo.
Furthermore, there has been no report with regard to the findings of IGF-II cDNA in tilapia (
Oreochromis mossambicus
), and the production of a biologically active IGF-II polypeptide from a tilapia cDNA.
The invention to be presented below describes the cloning and sequencing of a fish IGF-II gene and the production of the biologically active fish IGF-II recombinant polypeptides. The establishment of a gene expression system is important because it provides a multifaceted channel for studies of the functional activity of the expressed proteins. In this case, the successful development of a gene expression system for fish IGF-II is especially important because it not only allows for the production of high quality fish IGF-II protein which facilitates the antibody production for immuno-histochemical study (e.g., RIA, ELISA, etc.), but also provides large quantities of fish IGF-II protein for studies of the physiological functions of IGF-II in fish, particularly in determining the stimulatory effects on growth of juvenile fish.
The invention to be present below also discloses the findings concerning the mitogenic and anti-tumor effects of fish IGF-II E domain peptide. The findings are important because they may lead to major breakthrough in human growth and/or cancer prevention.
SUMMARY OF THE INVENTION
The present invention describes (1) the identification of the complete coding region of a fish genomic IGF-II in a cDNA library, (2) the sequence analysis of a fish IGF-II cDNA, (3) the construction of a recombinant fish IGF-II expression vector, (4) the expression of fish recombinant IGF-II peptides in
Escherichia coil
(
E. coli
), yeast, baculovirus, and fish cells, and (5) the biological functions of the fish recombinant IGF-II peptides.
The IGF-II B—D domain peptide, signal peptide, and E domain peptide can be isolated and purified from the host cells (e.g.,
Escherichia coli
, yeast, baculovirus, and fish cells) which have the capacity of expressing the vector comprising the IGF-II DNA sequence (SEQ ID NO:1), signal DNA sequence (SEQ ID NO:2), and E domain DNA sequence (SEQ ID NO:3), respectively. These peptides can also be produced by in vitro synthesis.
The functional studies of the E domain peptide (produced either from gene expression or in vitro synthesis) demonstrate that it possesses mitogenic and anti-tumor activities.


REFERENCES:
patent: 5476779 (1995-12-01), Chen et al.
DNA and Cell Biology J.Y. Chen et al. Production of Biologically Active Recombinant Tilapia Insulin-Like Growth Factor-II Polypeptides inEscherichia coliCell and Characterization of the Genomic Structure of the Coding Region 16:-883-892.
DNA Alane Gray et al. Tissue-Specific and Developmentally Regulated Transcription of the Insulin-Like Growth Factor 2 Gene vol. 6, No.4 1987.
Histo-chemistry Reinecke et al. IGF-2-like peptides are present in insulin cells of the elasmobranchian endocrine pancreas: an immunohistochemical and chromatographic study 102:365-371 (1994).
Proc. Natl. Acad. Sci. USA Michael Shamblott Identification of a second insulin-like growth factor in a fish species 89:8913-8917.
DNA and Cell Vadim Kavsan Structure of the Chum Salmon Insulin-Like Growth Factor I Gene 12:729-737 Biology.

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