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
2002-06-17
2004-12-07
Brumback, Brenda (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069400, C435S252100, C435S252330, C435S320100, C530S300000, C530S324000
Reexamination Certificate
active
06828124
ABSTRACT:
BACKGROUND OF THE INVENTION
In mammals, growth hormones (somatotropins) are initially expressed in a pre-hormone form comprising a leader sequence which is removed during secretion of the mature, biologically active hormone from pituitary cells. As used herein, “mature” somatotropin (“ST”) means having an amino acid length essentially like those of the ST secreted into the bloodstream of the animal, e.g., N-Ala-Phe-Pro- or N-Phe-Pro-bovine ST, N-Phe-Pro-human, -porcine (identical to canine) or -equine ST, etc.
Attempts to bacterially express human and bovine STs in non-secretion systems using structural genes having the sequences of their cDNAs were at first unsuccessful. Researchers succeeded after introducing silent mutations into the front end of the structural gene. P. H. Seeburg et al., attributed the original difficulties to translation being impeded by secondary structure of the mRNA corresponding to cDNA, and taught lessening such secondary structure to enable significant expression. DNA, Vol. 2, No. 1, 1983, pp. 37-45; U.S. Pat. Nos. 5,254,463 and 5,260,201.
It has been possible to obtain commercially viable ST expression levels using DNA containing certain silent mutations. However, the need for new vectors that are highly productive and less expensive to use has been difficult to satisfy because the relationships between the configurations and expression levels of “mature” somatotropin vectors remain poorly understood and highly unpredictable.
SUMMARY OF THE INVENTION
Described herein is a family of ST vectors with which micro-organisms, preferably bacteria such as, but not limited to
E. coli
, can be transformed to enable the expression of bovine ST (“bST”) at high levels using conventional fermentation and induction conditions. This family is exemplified by the 44 vectors listed in Table I each of which comprises the corresponding sequence of SEQ ID NOs 1 through 44, as indicated in Table I. Each of SEQ ID NOs 1 through 44, extends from an EcoRI site (GAATTC) before the promoter region, through the promoter, ribosome binding site (“RBS”) and entire bST structural gene, ending with the translation stop codon for the structural gene.
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Bogosian, G. et al. “Biosynthesis and incorporation into protein of norleucine byEscherichia coli,” J. of Biol. Chem., 264:531-539 (1989).
Seeburg, P.H. et al., “Efficient bacterial expression of bovine and porcine growth hormones.”DNA, 2:37-45 (1983).
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Aardema Noelle D.
Bogosian Gregg
O'Neil Julia P.
Bonner Grace L.
Brumback Brenda
Howrey Simon Arnold & White , LLP
Monsanto Technology LLC
Nichols Christopher James
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