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
1999-02-16
2002-02-26
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S477000, C536S023700
Reexamination Certificate
active
06350591
ABSTRACT:
FIELD OF THE INVENTION
The instant invention is related to the field of thermophilic microorganisms the stable gene transfection in and protein expression thereof, and in the genetic thermostabilization of proteins.
BACKGROUND OF THE INVENTION
Extreme thermophilic microorganisms such as Thermus, thrive in high-temperature environments that are lethal to other known forms of life. Fortunately, apart from their higher growth temperature requirement, they can be handled in the laboratory much like
E. coli
. Enzymes from thermophiles are thermostable and are therefore used in industrial processes that benefit from a high reaction temperature. Also, these enzymes have become widely used in molecular genetic research, for example, in the development and application of the polymerase chain reaction (PCR).
One area of particular interest in the field of thermophile research is the determination of molecular mechanisms underlying enzymatic thermostability. Ultimately, a better understanding of this phenomenon will allow mesophilic proteins to be rationally converted to thermostable proteins for industrial applications. Many groups have attempted to engineer thermostability into proteins through in vitro rational design approaches (Perry and Wetzel, 1984,
Science
226:555-557; Sauer, at al., 1986,
Biochemistry
25:5992-8; Pantoliano, et al., 1987,
Biochemistry
26:2077-82; Meng, et a/., 1993,
Bio/Technology
11:1157-1161) or through homology comparison and domain fusion of related proteins (Onodera, et al., 1991,
J Biochem
109:1-2; Barany, et al., 1992,
Gene
112:3-12; Politz, et al., 1993,
Eur J Biochem
216:829-34; Lee, et al., 1993,
J Bacteriol
175:5890-8). These approaches, however, often require either a three dimensional protein structure or a series of related proteins. For proteins which have not been well characterized, random mutagenesis can be a powerful tool if the proper selection or screen can be applied (Matsumura and Aiba, 1985,
J Biol Chem
260:15298-15303; Liao, et al., 1986,
Proc Natl Acad Sci USA
83:576-580; Kajiyama and Nakano, 1993,
Biochemistry
32:13795-9; Arnold, 1993,
Faseb J
7:744-9). The instant invention provides a genetic process for the insertion of exogenous protein coding sequences into, and direct selection of thermostable variants of mesophilic enzymes. Other “thermo-genetic” processes, were attempted by Liao (Liao et al., 1986,
Proc. Natl. Acad. Sci. USA
83: 576-580), EP Patent application 0 138 075, and by Matsumura (Matsumura et al., 1985,
J. Biol. Chem.
260: 15298-15303).
The concept of “thermo-genetics” consists of a method for introducing a gene of interest into a thermophile followed by a temperature-shift to select for temperature-resistant mutations in the corresponding protein of interest. The model thermo-genetic systems (EP Patent application 0 138 075; Matsumura, 1985; Liao, 1986) used the mesophilic kanamycin-resistance gene (kan) on a multicopy plasmid in the moderate thermophile
Bacillus stearothermophilus
. The kan gene was first introduced into
B. stearothermophilus
at the lowest permissible temperature of growth, 47° C. Two consecutive thermal shifts, first to 63° C. and then to 69° C., resulted in two corresponding thermo-stabilizing mutations, producing the double mutant allele, designated here as kan
tr2
. At this point the upper limit for permissible growth had been reached, creating a barrier to further selections for temperature-resistant mutations. Matsumura also performed a related series of experiments generating the same two mutants in parallel (Matsumura et al., 1986,
Nature
323: 356-358) and later showed they could be combined with an additive result.
While other host-vector systems have been developed for
Thermus thermophilus
, a closely related thermophile, they all have deficiencies in their ability to be used in a thermostabilization process and in stable integration of exogenous genes into Thermus. Two plasmid-based systems use multicopy plasmids with an unstable copy number which can interfere with mutant selection (Mather and Fee, 1992,
Appl Environ Microbiol
58:421-425; Lasa, et al., 1992,
J Bacteriol
174:6424-6431). The multicopy nature of these systems do not ideally lend themselves to thermostabilization of genes since many copies of the gene of interest are present, and can mask any desired mutations which may occur. In addition, reports with plasmid-based systems in Thermus indicate that the plasmids are very unstable, that copy number varies widely, and that gene duplication and amplification can occur, making them very difficult to use. Another approach which used an insertional mutagenesis system was developed by Lasa et. al. (Lasa, et al., 1992,
Molec Microbiol
6:1555-1564) but unfortunately caused a debilitating phenotype in the host organism.
In Lasa's insertional mutagenesis system, the kan
tr2
was inserted in single copy into a highly-expressed (slpA) region of the chromosome for use in chromosomal insertion strategy (Lasa et al. 1992a, J. Molec. Microbiol. 6, 1555-1564; Lasa et al. 1992b, J. Bacteriol. 174, 6424-6431). This system used the slpA gene which codes for an abundant cell surface protein and therefore was likely to be highly expressed. A high expression site was originally a logical choice for testing the feasibility of a single-copy system. Unfortunately, insertion into slpA results in debilitating growth and morphology phenotypes making it difficult to use the plasmid system.
References which define the background of the invention, but which are not necessarily prior art to the instant invention are as follows. The references cited herein above and below are hereby incorporated by reference in their entirety.
Sen & Oriel (1990) Transfer of transposon Tn916 from
Bacillus subtilis
to
Thermus aquaticus, FEMS Microbiology Letters
67:131-134, teach the use of the Streptococcus transposon Tn916, carrying tetracycline resistance for conjugal transfer into
Thermus aquaticus
via
Bacillus subtilis
. This was found to be effective at 48° C. and 55° C. The actual insertion site is unknown.
Koyama et al. (1990) A plasmid vector for an extreme thermophile,
Thermus thermophilus, FEMS Microbiology Letters
72:97-102, teach a Thermus-
E. coli
shuttle vector carrying a tryptophan synthetase gene (trpB). This cryptic plasmid pTT8, was able to transform
Thermus thermophilus
. The authors point out that a plasmid vector carrying trpBA was not suitable for selection since the cloned DNA fragment recombined with the chromosomal counterpart at high frequency.
Koyama & Furukawa (1990) Cloning and Sequence Analysis of Tryptophan Synthetase Genes of an Extreme Thermophile,
Thermus thermophilus
HB27: Plasmid Transfer from Replica-Plated
Escherichia coli
Recombinant Colonies to Competent
T. thermophilus
Cells,
J. of Bacteriology
72:3490-3495, disclose nucleotide sequences for trpBA genes, their use in plasmids and expression in
E. coli
under the control of the lac promoter.
Koyama et al. (1986) Genetic Transformation of the Extreme Thermophile
Thermus thermophilus
and of Other Thermus spp.,
J. of Bacteriology
166:338-340, discuss the conditions for optimal transformation with exogenous DNA. The use of
Thermus thermophilus
HB27 did not require chemical treatment to induce competence, although the addition of Ca
+2
and Mg
+2
was optimal. The optimal conditions were found to be 70° C. with a 60 minute incubation, pH 6 to 9.
Borges & Bergquist (1993) Genomic Restriction Map of the Extremely Thermophilic Bacterium
Thermus thermophilus
HB8,
J. of Bacteriology
175:103-110, teach the use of
Thermus thermophilus
HB8, which carries two cryptic plasmids, pTT8 and pVV8 was examined. A genomic restriction map was generated, 16 genes located on the map.
Matsumura et al. (1984) Enzymatic and Nucleotide Sequence Studies of a Kanamycin-Inactivating Enzyme Encoded by a Plasmid from
Thermophilic bacilli
in Comparison with That encoded by Plasmid pUB110
, J. of Bacteriology
160:413-420, teach the a Kanamycin resistance gene from a thermophilic bacteri
Casadaban Malcolm J.
Demirjian David C.
Pagratis Nikos C.
Vonstein Veronika
Weber J. Mark
Brusca John S.
McDonnell & Boehnen Hulbert & Berghoff
Thermogen Inc.
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