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Construction of Antitumor Strain

#1263


Construction of the Strain Overproducing the Recombinant L-Asparaginase Erwinia Carotovora and Elaboration of the Large-Scale Method for Purification of Gomogeneous Enzyme with High Antitumor Activity

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • BIO-IND/Industrial Biotechnology/Biotechnology

Status
8 Project completed

Registration date
10.04.1998

Completion date
03.10.2003

Senior Project Manager
Russo P A

Leading Institute
Institute of Biomedical Chemistry, Russia, Moscow

Supporting institutes

  • Research Center of Toxicology and Hygienic Regulation of Biopreparations, Russia, Moscow reg., Serpukhov

Collaborators

  • University of Arkansas for Medical Sciences, USA, AR, Little Rock\nTechnoLink Corp-82, USA, NJ, Clifford\nLoma Londa University / School of Medicine, USA, CA, Loma Linda\nWayne State University / School of Medicine, USA, MI, Wayne\nUS Food and Drug Administration / National Center for Toxicological Research, USA, AR, Jefferson

Project summary

The project is devoted to one of the actual problem of biochemistry, oncology and medicine, concerning with creation of a new effective preparations of L-asparaginase for cancer therapy. At present, microbial L-asparaginases are recognized as one of basic drugs for improvement of remission induction mainly in infant acute lymphoblastic leukemia and is being positively incorporated in the treatment of high risk diseases such as intractable acute lymphoblastic leukemia and the myelogenous recurrence. Up to now bacterial L-asparaginase is a single example of effective clinical using of enzymes in cancer therapy.

The choice of Erwinia carotovora L-asparaginase as the object of the present research project is explained its very highly specific activity and stability during of purification, existing only single therapeutically active isoform of enzyme, more strong antileukemic activity and less profound immunologic toxic effects comparatively with L-asparaginases from other bacteria.

The enzymes is typically produced by culturing microorganisms of the genus Escherichia or Erwinia in defined culture media. L-asparaginases are produced commercially as a therapeutic products in USA (Enzon), England (Porton), Germany (Bayer), Japan (Kyowa). Considerable effort has been devoted to the selection of strains of bacteria which are capable of producing L-asparaginase in high yields. However, even the best strains currently available (Er.carotovora SCR 193, Er.carotovora 268M, Er.chrysanthemi NCPB 1066) yield quantities of L-asparaginase which are lower than would be desirable.

This project relates to process for producing of L-asparaginase by culturing microorganisms containing genetic material produced by recombinant DNA techniques, purification of enzyme and analysis of its properties and antitumor activity.

The aim of the project is the construction by genetic engineering methods of the stable recombinant strain overproducing of Er.carotovora L-asparaginase with high enzymatic and antitumor activities of target enzyme, obtaining the novel mutant forms of the Er.carotovora L-asparaginase using the methods of rapid “molecular evolution” and selection, elaboration of effective purification methods for recombinant enzyme analogues with following preclinical trials of antilymphomic activity on cell lines and laboratory animals. These investigations can be considered as an initial step for creation of the novel recombinant medical preparation of L-asparaginase. The project goals can be achieved taking into account the significant experience of the applicant institutions in genetic engineering, protein chemistry, analytical and preparative biochemistry and functional analysis of biologically active preparations.

The main project goals:


· construction of the stable and efficient strain overproducing the recombinant Er.carotovora L-asparaginase in E.coli cells;
· application of the rapid “molecular evolution” methods in DNA shuffling experiments for the selection of novel recombinant analogues of this enzyme with useful enzymatic, biological and pharmacological properties;
· elaboration of methods for efficient large-scale purification of homogeneous (crystalline) recombinant Er.carotovora L-asparaginase and its mutant forms;
· comparison of the structure, physico-chemical and catalytic properties of the wild-type and recombinant analogues of L-asparaginase;
· studies of the specific antitumor activity, toxicity and pharmacokinetics of recombinant Er.carotovora L-asparaginase on the special cell lines and in experiments with laboratory animals.

The main expected results:


· with using of recombinant DNA technology a new high-level heterologous expression system of Er.carotovora L-asparaginase in E.coli cells would be constructed which may be useful for expression of L-asparaginases and asparaginase-glutaminases from other bacteria;
· on the basis of rapid evolution of the L-asparaginase in vitro by DNA shuffling method a novel recombinant analogues of this enzyme with useful enzymatic, biological and pharmacological properties would be obtained;
· a stable highly efficient strain overproducing the recombinant Er.carotovora L-asparaginase in E.coli cells would be obtained;
· efficient large-scale method for recombinant Er.carotovora L-asparaginase purification would be elaborated;
· new data about properties, structure and catalytic activity of recombinant forms of L-asparaginase would be obtained;
· recommendations for creation of pharmaceutical antitumor preparation the recombinant Er.carotovora L-asparaginase as well as to obtaining of modified (immobilized) forms of enzyme would be worked out.

Technical approach and methodology:

· The bacterial strain-overproducing the recombinant Er.carotovora L-asparaginase will be obtained by standard methods of molecular biology and genetic engineering including the construction of genomic library of Er. carotovora, PCR-amplification of the coding gene of Er.carotovora L-asparaginase based on the known sequences of highly homologous genes of L-asparaginases from other species of Erwinia, sequencing the cloned L-asparaginase gene and its subcloning into different E.coli expression vectors, including our own original expression systems.

· The creation of novel analogues of L-asparaginase will be achieved using the “molecular evolution” approach (W.Stemmer, 1995) in DNA shuffling experiments with subsequent selection of more potent enzyme forms based on the sensitive color plate assay for L-asparaginase.

· In order to purify recombinant L-asparaginase standard methods of protein chemistry and biochemistry will be used, including fractionation of macromolecules with organic solvents, pH- and thermal treatment, ion-exchange, affinity and adsorption chromatography, etc. The purified enzyme is also thought to be crystallized using organic solvents.

· Comparative study of structure, physico-chemical and catalytic properties of native and recombinant forms Er.carotovora L-asparaginase will be perfomed using methods of electrophoresis in polyacrylamide gel in native and denaturating conditions, isoelectrophocusing, chromatophocusing, gel-filtrarion, protein sequencing, kinetic analysis, etc.

· Investigation of antitumor activity of recombinant L-asparaginase (pharmacokinetics, acute and chronic toxicity, cumulation, specific antilymphomic activity, immunogenicity, etc.) will be performed using standard tests on the specific cell lines in vitro and in the experiments with the laboratory animals.

Potential role of foreign collaborators:

It is planned to provide joint investigations with foreign collaborators, scientific exchange of necessary preparations and information, combine publications, scientific visits to collaborators’ laboratories, seminars and consultations.

Up to date the following potential scientific collaborators have agreed to participate in joint research project to contribute in solving its separate tasks:


· Dr.Victor V.Adler, Ph.D., associate professor, Ruttenberg Cancer Center Mount Sinai, New-York, USA;
· Dr.Willem P.S.Stemmer, professor, Affymax Research Institute, Palo-Alto, USA;
· Dr.Vassilis Bouriotis, Ph.D., professor, Enzyme Technology Division IMBB, Heraklion, Crete, Greece.


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