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ELECTROPHORESIS OF PROTEINS:DATA CAPTURE,ANALYSIS AND CONSTRUCTION OF DATABANKS

Objective

IMPROVED AND MORE EFFICIENT TOOLS FOR THE SCREENING OF MICROBIAL STRAINS FOR BIOTECHNOLOGICAL PROCESSES, DETECTION OF CONTAMINANTS, IDENTIFICATION OF STRAINS, ESPECIALLY PATENTED BIOTECHNOLOGICAL AND PATHOGENIC STRAINS.

STUDY OF GENE EXPRESSION IN EUKARYOTE CELLS, UNDERSTANDING AND IMPROVED DIAGNOSIS OF DISEASE-RELATED PROTEIN ABNORMALITIES.
The technique for one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was standardised to enhance reproducibility, necessary for computer comparison, database construction and portability. The process was completely tested and the reproducibility obtained was sufficient to allow the possibility of large and long term database construction.

A Siemens 7570-C implemented FORTRAN 77 and UCSD-like PASCAL program has been developed for the storage, retrieval and modification of one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles. The database program was extended with procedures for both fast identification and numerical comparison of a limited set of 1-dimensional SDS-PAGE protein profiles (up to 800).

A microcomputer implemented PASCAL program was developed for the automated alignment of 1-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) protein profiles. The electrophoretic pattern of bacterial strain Psychrobacter immobilis LMG 1125 is used as a universal reference profile.

1-dimensional and 2-dimensional electrophoretic techniques provide powerful tools for the analysis of complex protein mixtures. Computer analysis of 1-dimensional and 2-dimensional protein patterns to establish databases of pathogenic bacteria, biotechnologically important bacteria and eucaryotic cells provides methods for screening and identifying bacterial strains, recognizing new groups, and characterizing human and animal disease related protein abnormalities. For wide use, standardization of techniques is of paramount importance.

A standard procedure has been developed for the production of 1-dimensional gels. A set of bacteria independently studied in 2 laboratories gave the same analytical result and the portability of the systems was demonstrated despite implementation on different computers using similar software. Databases were established for identification of bacterial species and protein types within species and standard 2-dimensional methodology was applied to human, murine, bacterial and streptomycete cells. A system using immobilized pH gradients for isoelectric focusing (IEF) was also developed providing greater reproducibility of 2-dimensional patterns. Computer systems of 2-dimensional gel analysis namely PDQUEST and BioImage were implemented and databases were established for the studied cell types. Standardized gels, analytical system and databases will allow major improvements in the study of developmental and disease processes in humans, and reliable, rapid identification of pathogenic and biotechnologically important bacteria.
DEVELOPMENT OF SOFTWARE AND DATABANK FOR 1-D PATTERNS OF BIOTECHNOLOGICALLY IMPORTANT BACTERIA, USING STRAINS HELD IN THE LABORATORY OF MICROBIOLOGY, GENT CULTURE COLLECTION (LMG), ON WHICH EXTENSIVE GENOMIC AND BIOTECHNOLOGICAL DATA EXISTS. THERE WILL BE CLOSE COLLABORATION WITH THE NATIONAL COLLECTION OF TYPE CULTURES (NCTC).COLINDALE, UK ON PREPARATION AND RUNNING OF GELS, EXCHANGE OF SOFTWARE FOR HARDWARE COMMON TO BOTH (LKB DENSITOMETERS/APPLE MICROCOMPUTERS), EXCHANGE OF STRAINS, EXTRACTS AND GELS. 2-D PAGE ON SELECTED STRAINS WILL BE ANALYSED IN COLLABORATION WITH THE ROYAL POSTGRADUATE SCHOOL, LONDON AND INSTITUTE PASTEUR, PARIS.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

RIJKSUNIVERSITEIT GENT
Address
Lab.voor Microbiologie Ledeganckstraat 35
9000 Gent
Belgium

Participants (5)

AND WITH THE COLLABORATION OF:
Central Public Health Laboratory
United Kingdom
Address
61 Colindale Avenue
NW9 5HT London
INSTITUT PASTEUR
France
Address
Rue Du Docteur Roux 25
75724 Paris
Joyce Loebl Ltd
United Kingdom
Address
Dukesway Team Valley Trading Estate
NE11 0PZ Gateshead
Royal Postgraduate Medical School
United Kingdom
Address
Du Cane Road
W12 0HS London