Following are the abstracts of those papers: As I mentioned the Patent TRCA calims as their novel method is a very simple method that has been used for years and is taught in every first year molecular biology textbook. As you can see, the first description of the same method as Patent 414 was published in 1985. Ther is no way this patent can hold. However, having said this, INSM needs to hire better lawyers, their preparation and defense during the last hearing was terrible.
1: J Mol Endocrinol. 1992 Jun;8(3):213-23.
Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I
indicate the relative importance of IGF-binding protein and receptor binding for
enhanced biological potency.
Francis GL, Ross M, Ballard FJ, Milner SJ, Senn C, McNeil KA, Wallace JC, King
R, Wells JR.
Cooperative Research Centre for Tissue Growth and Repair, CSIRO Division of
Human Nutrition, Adelaide, South Australia.
An efficient expression system in Escherichia coli for several biologically
active insulin-like growth factor-I (IGF-I) fusion peptide analogues is
described. These novel IGF-I fusion protein analogues have properties that make
them very useful reagents in the investigation of IGF-I action. The analogues
comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine
growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains
the authentic IGF-I sequence, and two analogues,
[Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I,
where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg
respectively. The three peptides are referred to as Long IGF-I, Long
[Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present.
Production of the purified fusion peptides was aided by folding the reduced and
denatured fusion peptide sequence under conditions that gave very high yields of
biologically active product. Introduction of a hydrophobic N-terminal extension
peptide appears to facilitate the correct folding of the IGF-I analogues
compared with that obtained previously when folding normal-length IGFs. The
biological activities of the IGF-I fusion peptides were compared with authentic
IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the
analogues were more potent than authentic IGF-I in their abilities to stimulate
protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells,
where the IGFs act through the insulin receptor, the Long IGF-I analogues
maintained a similar potency relative to IGF-I as was observed in the L6
myoblasts. The order of biological potency in cell lines secreting IGF-binding
proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater
than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken
embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the
medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor
and IGFBP association by these analogues reinforced our previous findings that
N-terminal analogues of IGF-I show increased biological potency due to changes
in the degree of their IGFBP interactions.
PMID: 1378742 [PubMed - indexed for MEDLINE]
2: J Mol Endocrinol. 1992 Feb;8(1):29-41.
Production and characterization of recombinant insulin-like growth factor-I
(IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu3,
following their expression in Escherichia coli as fusion proteins.
King R, Wells JR, Krieg P, Snoswell M, Brazier J, Bagley CJ, Wallace JC, Ballard
FJ, Ross M, Francis GL.
Department of Biochemistry, University of Adelaide, South Australia.
The development of an efficient expression system for insulin-like growth
factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The
fusion protein consists of an N-terminal extension made up of the first 46 amino
acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn.
The latter two residues provide a unique hydroxylamine-sensitive link between
[Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the
fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly
bond, refolding of the reduced IGF-I peptide and purification to homogeneity.
This expression system was also used to produce two variants of IGF-I in which
Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I
respectively. Production of milligram quantities of IGF-I peptide was readily
achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established
by high-performance liquid chromatography and N-terminal sequence analysis.
[Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays
measuring stimulation of protein synthesis and DNA synthesis or inhibition of
protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to
bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1
receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding
proteins rather than increased receptor binding is the likely explanation for
the greater biological potency of the analogues compared with IGF-I.
PMID: 1311930 [PubMed - indexed for MEDLINE]
3: Methods Enzymol. 1991;198:3-16.
Expression and purification of recombinant insulin-like growth factors from
Escherichia coli.
Nilsson B, Forsberg G, Hartmanis M.
PMID: 1857223 [PubMed - indexed for MEDLINE]
4: Eur J Biochem. 1989 Apr 1;180(3):555-61.
Gene synthesis, expression in Escherichia coli and purification of
immunoreactive human insulin-like growth factors I and II. Application of a
modified HPLC separation technique for hydrophobic proteins.
Hummel M, Herbst H, Stein H.
Institute of Pathology, Klinikum Steglitz, Free University of Berlin.
We have expressed synthetic genes encoding human insulin-like growth factors I
and II in large quantities in Escherichia coli as fusion proteins with the 300
N-terminal amino acids of the E. coli trpE gene product. The resulting hybrid
proteins were purified from the insoluble fraction of crude bacterial lysates
using a rapid HPLC separation procedure employing a C8 reversed-phase column and
a gradient of 2-propanol in formic acid. With the procedure we obtained in
volatile solvents highly purified fusion proteins that were used for further
biochemical and immunological procedures. Here we describe biochemical
characteristics of the bacterially expressed fusion proteins and demonstrate
that these proteins share substantial antigenic properties with the native
polypeptides allowing the establishment of highly specific monoclonal
antibodies.
PMID: 2469575 [PubMed - indexed for MEDLINE]
5: Gene. 1988 Sep 7;68(2):193-203.
Expression of secreted insulin-like growth factor-1 in Escherichia coli.
Wong EY, Seetharam R, Kotts CE, Heeren RA, Klein BK, Braford SR, Mathis KJ,
Bishop BF, Siegel NR, Smith CE, et al.
Department of Biological Sciences, Monsanto Co., Chesterfield, MO 63198.
The synthesis, processing and secretion of insulin-like growth factor-1 (IGF-1
or somatomedin-C) fused to LamB and OmpF secretion leader sequences in
Escherichia coli have been investigated. Expression and secretion of IGF-1 was
achieved. The major portion of this secreted IGF-1 accumulated in the
periplasmic space as insoluble aggregates. A small amount of IGF-1 was found
folded in its native conformation in the medium. The lamB and ompF signal
sequences were fused to the 5' coding sequence of IGF-1. Fusion of the lamB
signal sequence directly to IGF-1 (lamB-IGF-1) resulted in accumulation of 16-20
micrograms/A550/ml of correctly processed IGF-1 in the periplasmic space. The
processing efficiency of LamB-IGF-1 and OmpF-IGF-1 was enhanced in an E. coli
strain bearing a prlA4 mutation. Amino acid sequence analysis of IGF-1 secreted
into the periplasm and exported into the medium confirmed the precise removal of
the LamB or OmpF signal sequence. IGF-1 synthesized in E. coli was demonstrated
to be active in a cell proliferation bioassay.
PMID: 3065142 [PubMed - indexed for MEDLINE]
6: Biochemistry. 1987 Aug 25;26(17):5239-44.
Expression of human insulin-like growth factor I in bacteria: use of optimized
gene fusion vectors to facilitate protein purification.
Moks T, Abrahmsen L, Holmgren E, Bilich M, Olsson A, Uhlen M, Pohl G, Sterky C,
Hultberg H, Josephson S, et al.
Department of Biochemistry, Royal Institute of Technology, Stockholm, Sweden.
Several fusions between the gene for human insulin-like growth factor I (IGF-I)
and the genes for different IgG-binding fragments of staphylococcal protein A
were assembled and compared regarding expression, secretion, and purification of
the peptide hormone. After IgG affinity purification of the fusion proteins from
the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was
released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized
expression system based on a modified synthetic IgG-binding domain (z),
resistant to hydroxylamine, gave the highest yield of fusion protein. After
cleavage, the hormone could be separated from the IgG-binding moiety and from
noncleaved fusion protein by a second passage through the IgG affinity column.
The biological activity and the purity of the IGF-I obtained were confirmed by a
radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel
electrophoresis, isoelectric focusing, and high-performance liquid
chromatography.
PMID: 3676250 [PubMed - indexed for MEDLINE]
7: Nucleic Acids Res. 1985 Mar 25;13(6):1923-38.
Optimizing the expression in E. coli of a synthetic gene encoding somatomedin-C
(IGF-I).
Buell G, Schulz MF, Selzer G, Chollet A, Movva NR, Semon D, Escanez S, Kawashima
E.
Double-stranded DNA encoding the human hormone somatomedin-C (SMC) has been
synthesized. This synthetic gene has been inserted into a plasmid bearing the
strong leftward promoter (PL) of bacteriophage lambda and expressed in E. coli.
Codons for the N-terminal region of SMC which maximized the hormone's synthesis
were chosen in an SMC-lac z fusion assay. The amounts of SMC accumulated in E.
coli were influenced by mutations at two chromosomal loci, lon and htpR.
