Cloning, functional expression in Pichia pastoris, and purification of potato cystatin and multicystatin.
In the tubers and leaves of potato, Solanum tuberosum, cysteine protease inhibitors are thought to play roles in the defence against herbivores and in regulating physiological processes like senescence and cell death. The cDNAs for two such inhibitors, potato multicystatin (PMC) with 8 cystatin domains and potato cystatin (PC) with a single domain, were cloned and expressed in the yeast Pichia pastoris. PC yielded on average 100 mg of purified active protein from 1l of culture supernatant. Purification to homogeneity was done in one step by cation exchange. The apparent equilibrium dissociation constant (K(i)) for papain was 0.1 nM. Cloning of the PMC cDNA was successful despite apparent toxicity for Escherichia coli and a high frequency of recombination events in RecA- strains of E. coli. In yeast, the expression of the cloned full length PMC gene was poor compared to that of the single domain.
Annadana
Seetharam
S
Business Unit Cell Cybernetics, Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands.
Schipper
Bert
B
Beekwilder
Jules
J
Outchkourov
Nikolay
N
Udayakumar
Makarla
M
Jongsma
Maarten A
MA