Production of bovine growth hormone by microorganisms

Details Production of bovine growth hormone by microorganisms Production of bovine growth hormone by microorganisms

1995-06-01

2001-05-08

Saoud, Christine (Department: 1647)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S069400, C435S252800, C435S320100, C530S399000

Reexamination Certificate

active

06229003

ABSTRACT:

FIELD OF INVENTION
The invention relates to novel modified microorganisms adapted to produce a polypeptide which has the bovine growth hormone amino-acid sequence. The invention further relates to a method for the modification of easily cultivable non-pathogenic microorganisms so as to convert same to effective producers of bovine growth hormone containing polypeptide.
The invention further relates to the production of bovine growth hormone by means of easily cultivable non-pathogenic microorganisms.
BACKGROUND OF THE INVENTION
DNA which has the capacity to code for polypeptides, can be synthesized and spliced into plasmid DNA and then when cells are transformed by this recombinant DNA, the synthetic DNA will replicate in these cells. If properly inserted in phase with a suitable bacterial promoter, the synthetic DNA will be decoded (expressed) into the authentic polypeptide. The technique can be used for the production of various hormones, enzymes and other polypeptides. In each case the technique has to be adapted to the specific circumstances.
SUMMARY OF THE INVENTION
The present invention relates to novel modified microorganisms adapted to produce bovine growth hormone. The invention further relates to a process for the production of bovine growth hormone by the cultivation of such novel microorganisms. The invention further relates to the production of bovine growth hormone which comprises extracting RNA from bovine pituitaries, transcribing double-stranded DNA on these RNA templates after tailing the DNA with oligo-deoxycytidine, ligating the tailed plasmid DNA, selecting bacteria colonies containing the recombinant plasmids and cultivating said colonies to obtain a culture of cells containing the desired polypeptide and extracting same from such cultures.
According to a preferred embodiment of the invention, there are provided modified
E. coli
strains adapted to produce the desired growth hormone. Preferably, the strain used is one derived from
E. coli
HB101, and a preferred plasmid used is pBR322.
DNA was synthesized enzymatically on mRNA isolated from bovine pituitaries. The DNA was spliced into the beta-lactamase gene of a commonly used plasmid, pBR322
. E. coli
cells, preferably of the BB101 strain, or of similar strains, were transformed by the recombinant plasmids and colonies containing these plasmids were isolated. Colonies (clones) containing the coding capacity for bovine growth hormone and colonies containing the coding capacity for prolactin were identified by DNA sequencing. The inserted DNA from a colony containing the full length coding sequences for growth hormone was isolated and reinserted into another plasmid in a position adjacent to the beta-galactosidase promoter. Several colonies producing a polypeptide, which was characterized to be the desired growth hormone by gel electrophoresis and by precipitation with antiserum raised in response to pure authentic bovine growth hormone were isolated.
Procedure Used for the Construction of the Bacterial Strain which has the Capacity to Produce the Bovine Growth Hormone Fused to a Beta-lactamase Fraction
RNA from a pool of several calf pituitary glands was extracted by phenol and the poly(A)-containing RNA purified by chromatography on oligo(dT)-cellulose. The poly(A)+RNA was transcribed into double-stranded DNA by reverse transcriptase (AMV). The DNA was treated with single-stranded specific nuclease (S1) and dC-tailed by terminal transferase (PL Biochemicals) and then separated by electrophoresis on an agarose gel. The higher molecular weight fraction above 500 base-pairs was isolated and annealed to dG-tailed Pst 1 cut PBR322 plasmid DNA which was prepared by the standard procedure. Ca++ treated
E. coli
HB101 was exposed to this DNA preparation. Tetracycline resistant colonies were isolated and screened for recombinant plasmids by in situ hybridization with
32
P-labeled cDNA reverse transcribed from the poly(A)
+
RNA fraction mentioned above. Positive colonies were isolated and further screened for plasmids containing DNA sequences corresponding to bovine prolactin and growth hormone in the following way: Plasmids were prepared from each colony, the DNA was immobilized onto nitrocellulose filters and hybridized to Poly(A)
+
RNA extracted from pituitaries. The hybridized RNA was analyzed by its capacity to elicit the synthesis of a specific band corresponding to bovine growth hormone in a cell-free translation system. Most of the colonies were thus identified to contain DNA sequences coding for prolactin; a few have been identified to contain DNA coding for growth hormone. This was confirmed by determination on the part of the DNA sequences in an example from each group. The capacity to synthesize a polypeptide corresponding to growth hormone was analyzed by immunological techniques in the following way: The cells were labelled with
35
S-methionine for 30 minutes and the proteins extracted, then incubated with an antiserum raised against purified bovine growth hormone, followed by incubation with chloroform treated Staphilococcus A. The precipitated antibody-antigen complex was then dissociated by sodium dodecysulfate, beta-mercaptoethanol and analyzed by gel electrophoresis. Several colonies were found to synthesize a polypeptide which specifically reacted with this anti-serum. The size of the growth hormone polypeptide varied; in one strain the main polypeptide was about 40K dalton, in another strain its size was about 25K. The amount synthesized in a liter fermentation vessel was determined by radioimmunoassay as described below. The strain producing the highest yield of bovine growth hormone polypeptide was designated strain D
4
, deposited in the American Type Culture Collection, Rockville, Md., as ATCC No. 31826.
DNA Splicing to Obtain a Series of Modified Bovine Growth Hormones
construction of plasmids which produce modified bovine growth hormone (bGH) in bacteria
E. coli
is presented.
All constructions are based on the DNA fragment obtained from the DNA of pBR322 plasmid that harbors the DNA for bGH, i.e., the DNA from strain D
4
. The fragment is generated by cleavage of this plasmid obtained from strain D
4
with Hae II restriction endonuclease and will be designated fragment I. Fragment I comprising 1640 base pairs starts with the nucleotide No. 3 encoding the mature bGH, continues through all sequences.of bGH DNA including termination codon, then traverses through pBR322 DNA sequences from nucleotide 3612 through 2721 according to the published sequence of Sutcliffe (1978) Nucleic Acid Res 5, 2721. Therefore the beginning of fragment I is as follows:
       Phe
2
 Pro
3
 Ala
4
 Met
5

   C   TTC  CCA  GCC  ATG......

CGCGG  AAG  GGT  CGG  TAC......
and is missing two nucleotides necessary to encode the first amino-acid Ala.


REFERENCES:
patent: 2370154 (1945-02-01), Fleischer et al.
patent: 3886132 (1975-05-01), Brewer et al.
patent: 4010148 (1977-03-01), Goldstein
patent: 4342832 (1982-08-01), Goeddel et al.
patent: 2535554 (1974-08-01), None
patent: 0012494 (1979-08-01), None
patent: 1568047 (1978-05-01), None
Seeburg et al., Nature 276:795-798 (1978).
Lingappa et al., Cell Biol. 74:2432-2436 (1977).
Cohen et al., P.N.A.S. 70 (11):3240-3244 (1973); and.
Chang et al., P.N.A.S. 71 (4):1030-1034 (1974).
Nilson et al. (1979), “Purificatio of Pre-prolactin mRNA from Bovine Anterior Pituitary Glands”, J. Biol. Chem. 254:1516-1520.
Keiichi Itakura and Arthur D. Riggs (1980), “Chemical DNA Synthesis and Recombinant DNA Studies”, Science 209:1401-1405.
Hunt and Dayhoff (1976), Atlas of Protein Sequence and Structure, (National Biomed. Res. Fund.) Washington, D.C., vol. 5, Supp. 2, p. 11.
Edge et al. (1981), “Total Synthesis Of A Human Leukocyte Interferon Gene”, Nature 292:756-762.
Coutelle et al. (1978), “Use of Matrix-Immobilised Recombinant Plasmids To Purify Chain-Specific Rabbit Globin Complementary DNAs”, Ge

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