Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1998-08-07
2001-09-11
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S471000, C435S252330
Reexamination Certificate
active
06287866
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The subject invention relates to &bgr;-casein expressing constructs which have significant stability when introduced into host cells. These constructs, for purposes of the present invention, have been designated as pRAB-84-69 and pRSB-14.
2. Background Information
Stability of a plasmid in an induced cell culture is influenced by the external physical culture conditions as well as the internal environment of the cell. There are several external factors that may have an effect on plasmid stability. For example, it has been shown that a lack of carbon, nitrogen, phosphate and minerals can negatively affect plasmid stability (Godwin et al.,
J. Gen. Microbiol
. 111:201-10 (1979)). Use of a richer media may therefore alleviate plasmid loss in some cases.
The physiology of the host strain is another factor which has an important effect on plasmid stability, as the same plasmid can show different rates of loss depending on the host (Caulcott et al.,
J. Gen. Microbiology
133:1881-89 (1987)). Some plasmids are inherently unstable due to improper replication and segregation. Unstable segregation in the earlier stages of the culture gives a growth disadvantage to the cells harboring the plasmid, consequently leading to an increased proportion of cells without the plasmid. Maintenance of the plasmid in the culture also requires optimum levels of selective pressure on the culture to ensure that only cells harboring the plasmid with the selectable marker survive.
In addition to the above mentioned factors, the absence or presence of specific sequences may also affect plasmid stability. Chiang and Bremmer studied the stability of plasmid pBR322 and its tet, bla and rom derivatives (Chiang et al.,
Plasmid
20:207-220 (1988)). They reported that transcription of tet sequences present on pBR322 affects cell viability and may reduce plasmid stability. In other instances, the presence of certain sequences such as the par stability locus has been shown to stabilize plasmids with segregational instability (Austin et al.,
Plasmid
20:1-9 (1988)). Furthermore, the size of the inserted DNA and the “act of introducing” foreign DNA are also known to effect plasmid stability. Stability is negatively affected by inserts over 8 kb in size and has been attributed to replication fidelity, segregation or low copy number (Warnes et al.,
Plasmid
16:116-23 (1986)).
Such instability prevents large-scale or efficient production of the protein encoded by the nucleotide sequences present in the plasmid. Thus, such instability may have quite a large impact on the production level of the protein as well as on the development of diagnostic and therapeutic products. Additionally, genetic instability of the plasmid is important with respect to the receipt of regulatory approval for a commercial process.
The present constructs, derived from pRJB36, are quite stable and therefore permit the expression of large quantities of the desired end product. For example, the constructs may be used in the expression of a protein such as recombinant human &bgr;-casein. Thus, the present constructs overcome many of the disadvantages associated with known constructs such as pRJB36.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.
SUMMARY OF THE INVENTION
The present invention includes an isolated DNA sequence comprising a nucleotide sequence encoding a protein, wherein the nucleotide sequence is operably linked to a promoter, a nucleotide sequence encoding a first subunit of a kinase, a nucleotide sequence encoding a second subunit of the kinase, a nucleotide sequence encoding a peptidase and a nucleotide sequence encoding a bacterial resistance marker. The protein may be, for example, a human milk protein such as recombinant human &bgr;-casein, a protein from an edible plant, an antibody and an antigen. The promoter may be selected from the group consisting of Ptac, Pgal, T7, &lgr;P
L
, &lgr;P
R
, bla and spa. The kinase may be, for example, casein kinaseII&bgr;&agr;, and the peptidase may be, for example, aminopeptidase and iminopeptidase. The bacterial resistance marker may be selected from the group consisting of ampicillin resistance, kanamycin resistance, chloramphenicol resistance, and tetracycline resistance. The isolated DNA sequence may further comprise a transcriptional terminator such as rrnBT1T2.
Additionally, the present invention also encompasses a vector containing the isolated DNA sequence described above as well as a host cell containing this vector. The host cell may be prokaryotic, for example, a bacterial cell, such as Escherichia spp. (e.g.,
E. coli
) or eukaryotic.
Furthermore, the present invention also includes a method of producing a protein comprising introducing a vector into a host cell under time and conditions sufficient for expression of the protein. The vector may comprise an isolated DNA sequence comprising i) a nucleotide sequence encoding a protein, wherein the nucleotide sequence is operably linked to a promoter, ii) a nucleotide sequence encoding a first subunit of a kinase, iii) a nucleotide sequence encoding a second subunit of a kinase, iv) a nucleotide sequence encoding a peptidase and v) a nucleotide sequence encoding a bacterial resistance marker. The isolated DNA sequence may also comprise a transcription terminator such as rrnBT1T2. The protein, kinase, peptidase, promoter and bacterial resistance marker may be as described above.
Additionally, the present invention includes a pharmaceutical or nutritional composition comprising a protein produced according to the method described above.
The present invention also includes a vaccine which comprises a protein produced in accordance with the method described above.
Moreover, the present invention also encompasses a method of improving the genetic stability of a plasmid-containing cell during fermentation. This method comprises the step of transforming a cell with the above described vector, prior to fermentation, growing the cell in a culture utilized for inoculating a fermentor, growing the cell in the fermentor, inducing at least one of the nucleotide sequences present in the vector, and completing fermentation under for a time and under conditions suitable for optimal expression of the induced nucleotide sequence or sequences.
REFERENCES:
patent: 4543329 (1985-09-01), Daum et al.
patent: 5013662 (1991-05-01), Ben-Bassat et al.
patent: 5506209 (1996-04-01), Mukerji et al.
patent: 5538952 (1996-07-01), Mukerji
patent: 5710044 (1998-01-01), Mukerji et al.
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Greenberg et al.,Journal of Biological Chemistry, 259:5132-38.
Hanson et al,.Protein Expression and Purification, 4:373-381 (1993).
A. B-Bassat et al.,Journal of Bacteriology, 169:751-757 (1987).
Lingappa et al.,Proceeding of the National Academy of Science USA, 74:2432-2436 (1977).
Hitzeman et al.,Science, 219:620-25 (1983).
Klein et al.,Microbiology(1994) 140, 1133-1139.
Leenhouts et al.,Applied and Environmental Microbiology, 55:2, 394-400 (1989).
Johnson et al.,Bio/Technology, 12:1357-1360 (1994).
Mayo et al., “Applied and Environmental Microbiology” Jan. 1991 p. 38-44.
A B-Bassat et al.,Purification and Analysis of Recombinant Proteins eds. Seetharam and Sharma, pp. 147-159, Marcel Dekker Inc., N.Y. (1991).
J. M. Thurmond et al., “Expression And Characterization Of Phosphorylated Recombinant Human B-Casein InEscherichia Coli”—Vol. 10, 1997, pp. 202-208, Protein Expression and Purication.
Shen T.-J. et al.,Proceedings of the National Academy of Sciences of USA, vol. 90, Sep. 1993, pp. 8108-8112 —XP002058672.
Sabin E. A. et al., “High -Level Expression And In Vivo Processing of Chimeric Ubiquitin Fusion Proteins InSaccharomyces Cerevisiae”, vol. 7, No. 7, Jul. 1989, pp. 705-709, XP00
Chaudhary Sunita
Lemmel Steven A.
Leonard Amanda Eun-Yeong
Mukerji Pradip
Abbott Laboratories
Becker Cheryl L.
Nashed Nashaat T.
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