Genetically engineered glutaminase and its use in antiviral and anticancer therapy
a technology of antiviral and anticancer therapy, which is applied in the direction of depsipeptides, peptide/protein ingredients, fusion polypeptides, etc., can solve the problems of unsuitable therapeutic use of known mammalian glutaminase enzymes, unprofitable therapeutic use of glutaminases (a and b), and no therapeutically useful glutaminases available cheaply, so as to reduce the production cost of glutamina
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
example 1
[0074] This example demonstrates the identification of a clone containing the sequence coding for Pseudomonas 7A glutaminase and determination of its nucleotide sequence.
[0075] The glutaminase product is an enzyme that degrades glutamine (an amino acid that participates in more metabolic processes than any other amino acid) and therefore hinders growth. In more than two dozen independent experiments, we had been unable to clone the glutaminase in a variety of contexts. We have found it unclonable in high copy number backgrounds such as pUC. It also proved refractory to cloning in the absence of an upstream transcriptional terminator and a very tightly regulated promoter.
[0076] Chromosomal DNA was isolated from Pseudomonas 7A (a soil isolate organism, which has been deposited with the American Type Culture Collection under deposition number ATCC 29598) essentially as described (Strom, 1986, J Bacteriol. 165:367-372), and was partially digested with the restriction enzyme Sau3A. Fra...
example 2
[0082] This example demonstrates the expression of the gene for Pseudomonas 7A glutaminase.
[0083] Initial experiments showed that even among strong, regulated promoters (e.g, λP_PGA was refractory to overproduction. In order to obtain high level controlled expression of the Psuedomonas 7A (P7A) glutaminase in Escherichia coli, we first designed a new vector, pME15 (see FIGS. 3A and 3B for cloning, and Table 2). The backbone of the vector was pME12 (see Table 2) and contains the following features: β-lactamase gene (conferring ampicillin resistance), lac I (repressor of the lactose operon), a T7 transcriptional terminator, and a low copy-number ColE1-type origin of replication (pBR322-derived).
TABLE 2Plasmids Used in Constructionof a High-Level Expression PlasmidpME0.5 -genomic clone from a library of Sau3A fragmentsof P7A chromosomal DNA cloned into theBamHI site of pBR322. This clone contains thefull-length glutaminase gene.pME1 -the N-terminal 1.1 kb SalI fragment of pME0.5clon...
example 3
[0091] This example demonstrates the use of P7A glutaminase sequences to identify homologous sequences in other bacterial species.
[0092] Chromosomal DNA from Pseudomonas aeruginosa and Achromobacter sp. was isolated using standard protocols. After complete digestion with EcoRI, DNA fragment were resolved on a 30 cm, 1% agarose gel at 50 V for 15 hours in 89 mM Tris-Cl, pH8;89 mM Borate; and 1 mM EDTA. Transfer and hybridization were as described (Maniatis et al. Molecular cloning: A laboratory Manual, pp. 382-389, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1982) using stringent conditions. The probe was the coding region of the P7A glutaminase gene labeled with α-32P deoxycytosine triphosphate. Lane 1, Pseudomonas 7A (2 hr. exposure); lane 2, Pseudomonas aeruginosa (6 hr. exposure); lane 3, Achromobacter sp. (24 hr. exposure). Results are shown in FIG. 6.
PUM
| Property | Measurement | Unit |
|---|---|---|
| pH | aaaaa | aaaaa |
| pH | aaaaa | aaaaa |
| concentration | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More