Biochemical and molecular characterisation of Tetrahymena thermophila extracellular cysteine proteases.
BACKGROUND
Over the last decades molecular biologic techniques have been developed to alter the genome and proteome of Tetrahymena thermophila thereby providing the basis for recombinant protein expression including functional human enzymes. The biotechnological potential of Tetrahymena has been proved in numerous publications, demonstrating fast growth, high biomass, fermentation in ordinary bacterial/yeast equipment, up-scalability, existence of cheap and chemical defined media. For these reasons Tetrahymena offers promising opportunities for the development of a high expression system. Yet optimised high yield strains with protease deficiency such as commonly used in yeast and bacterial systems are not available.


RESULTS
This work presents the molecular identification of predominant proteases secreted into the medium by Tetrahymena thermophila. A one-step purification of the proteolytic enzymes is described.


CONCLUSIONS
The information provided will allow silencing of protease activity by either knock out methods or by Tetrahymena specific antisense-ribosome-techniques. This will facilitate the next step in the advancement of this exciting organism for recombinant protein production.
Herrmann
Lutz
L
Cilian AG, Johann-Krane-Weg 42, Muenster D-48149, Germany. herrmann@cilian.de
Erkelenz
Michael
M
Aldag
Ingo
I
Tiedtke
Arno
A
Hartmann
Marcus W W
MW
D000595:Amino Acid Sequence; D000818:Animals; D001709:Biotechnology / Q000379:methods; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D018503:Genome, Protozoan; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D016415:Sequence Alignment; D016808:Tetrahymena thermophila / Q000201:enzymology* / Q000235:genetics
D015853:Cysteine Proteinase Inhibitors; D015800:Protozoan Proteins; D003546:Cysteine Endopeptidases