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Coding calicheamicin biosynthesis and micromonospora echinospora gene for same antagonism

A biosynthetic technology of Micromonospora aculeatus, applied in the field of isolating genes and their corresponding proteins, can solve problems such as lack of knowledge

Inactive Publication Date: 2006-04-26
SLOAN KETTERING INST FOR CANCER RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prior to this discovery by the present inventors, there was a complete lack of knowledge about the genes encoding the biosynthesis of the non-pigmented protein enediyne

Method used

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  • Coding calicheamicin biosynthesis and micromonospora echinospora gene for same antagonism
  • Coding calicheamicin biosynthesis and micromonospora echinospora gene for same antagonism
  • Coding calicheamicin biosynthesis and micromonospora echinospora gene for same antagonism

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] To elucidate nucleotide sequences as quickly as possible, thermal cycle sequencing was performed from pUC- or pBluescript-based subclones (using M13 primers and primer walking), but also directly from isolated cosmids (via primer walking). Nucleotide sequence data were obtained using two Applied Biosystems Auto 310 Genetic Analyzers, followed by Applied Biosystems AutoAssembler TM DNA sequence assembly software for assembly. Dear, S. et al., Nucl Acids Res., 14, 3907-3911 (1991); Huang, X., Genomics, 14, 18-25 (1992). The Orf arrangement was calculated using the calculation program MacVector TM Combination of 6.0 and Brujene. MacVector is a commercially available software package that has the ability to construct a Micromonospora codon bias table (from known Micromonospora sequences) and then use this codon bias table to retrieve optimal orfs. Fickett, J.W., Nucleis Acids Research, 10, 5303-5318 (1982). Alternatively, the trialware program Brujene is specially des...

Embodiment 2

[0108] Example 2: Isolation and characterization of calC

[0109] cosmid or plasmid

The maximum tolerated concentration of calicheamicin

Cosmids 3a, 4a, 10a, 13a and 16a

0.5 μg ml -1

pJT1214 to pJT1232

5.0 μg ml -1

PRE7

20.0 μg ml -1

pRE7

50.0 μg ml -1

pJT1224, pAP6, Pre1, and control plasmids

pUC18, pBluescript, and pMALC2

-1

[0110] Nucleotide sequence analysis of the PstI-SacI fragment revealed that it contains two possible orfs. The proximal 1 kb of the fragment carries a single orf calD, while the distal 1 kb has orf calC. In silico translation of CalC and subsequent BLAST analysis revealed no homology to known proteins, whereas translation of calD to its protein CalD revealed the presence of three amino acid motifs that are normally expressed in O-methyl transfer The enzyme's S-adenosyl nucleosyl group is conserved in methionein. Therefore, it was assumed that calD was not resp...

Embodiment 3

[0112] Embodiment 3: the expression of protein CalC

[0113] The protein mbp-CalC was overexpressed and purified for further analysis. mbp-CalC was purified to homogeneity from pRE7 / E. coli as identified by SDS-PAGE. Overnight LB culture derived from fresh pRE7 / Escherichia coli colonies (containing 50 mg ml -1 Ampicillin and 50ng ml -1 Calicheamicin) grow at 37°C, 250rpm to A 600 = 0.5, induced with 0.5 mM IPTG, and continued to grow overnight. Cells were harvested (4,000 xg, 4°C, 20 min), resuspended in buffer A (50 mM Tris-Cl, pH 7.2, 200 mM NaCl, 1 mM EDTA), and sonicated. Cell debris was removed by centrifugation (5,000 xg, 4°C, 20 minutes). Apply the supernatant to an amylose affinity column (1.5×7.0cm, 1mL min -1 )superior. The desired mbp-CalC protein was eluted with buffer A containing 10 mM maltose. The eluate was concentrated and chromatographed on an S-300 column (50 mM Tris-Cl, pH 7.5, 200 mM NaCl). The active fraction was used immediately, or stored froze...

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Abstract

An isolated gene cluster of Micromonospora echinospora which codes for calicheamicin biosynthesis. The biosynthetic gene cluster contains genes encoding proteins and enzymes used in the biosynthetic production of calicheamicin, including the aryltetrasaccharide and aglycone. The gene cluster also includes the gene coding for the protein conferring calicheamicin resistance. The invention also provides isolated genes of the biosynthetic cluster and their corresponding proteins. In addition, the invention relates to DNA hybridizing with the calicheamicin gene cluster and the isolated genes of that cluster. Expression vectors containing genes of the biosynthetic gene and their functional variants are also provided. The invention also relates to host cells conjugated with DNA isolated from the Micromonospora echinospora spp. calichensis genome.

Description

[0001] This application is a PCT application of U.S. application U.S. 09 / 724,797, filed November 28, 2000, which is a continuation-in-part of U.S. application U.S. 09 / 457,045, filed December 7, 1999, and claims priority thereof right, which application is hereby incorporated by reference in its entirety. This application also claims priority to provisional application 60 / 111,325, filed December 7, 1998, which is hereby incorporated by reference in its entirety. field of invention [0002] The present invention relates to a biosynthetic gene cluster of Micromonospora echinospora spp. calichensis. In particular, a calicheamicin biosynthetic gene cluster containing genes encoding proteins and enzymes in the biosynthetic pathway and configuration of the aryltetrasaccharides and aglycones of calicheamicin, and which confers an increased Richeamicin resistance. The invention also relates to isolated genes of the biosynthetic clusters and their corresponding...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/09A61K31/7048A61P35/00A61P35/02C07H17/08C07K14/195C07K14/36C12N1/21C12N15/52C12P17/00C12P19/56C12P19/62C12P19/64
CPCA61P35/00A61P35/02C07K14/36C12N9/00C12N15/52C12P17/00C12P19/56C12P19/62C12P19/64
Inventor J·托尔森
Owner SLOAN KETTERING INST FOR CANCER RES
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