Expression, purification, and characterization of equistatin in Pichia pastoris.
Equistatin (EI) is a cysteine protease inhibitor that was isolated from the sea anemone Actinia equina. It belongs to a recently discovered group of thyroglobulin type-I domain inhibitors called thyropins. Since native EI is found only in low amounts in the body of sea anemone and expression of recombinant EI in Escherichia coli yielded only 1 mg/liter of protein, we used the Pichia pastoris expression system to obtain higher yields. A cDNA encoding EI was inserted into pPIC9 vector and transformed into the P. pastoris, strain GS115. Clones expressing high levels of EI were selected from 48 transformants. Recombinant EI was produced in 2-liter shake flasks and recovered from the fermentation broth by affinity chromatography using CM-papain-Sepharose. SDS-PAGE and N-terminal sequence analysis revealed that EI was N-terminally intact and running at the expected molecular weight of 22 kDa. The equilibrium dissociation constants of EI with papain and bovine cathepsin D were determined and were found to be similar to the results for the native inhibitor. EI production was scaled up to a bench top fermentor with a 25 mg/liter yield of active EI.
Rogelj
B
B
Jozef Stefan Institute, Jamova 39, Ljubljana, SI-1000, Slovenia.
Strukelj
B
B
Bosch
D
D
Jongsma
M A
MA
D019149:Bioreactors; D015153:Blotting, Western; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005822:Genetic Vectors; D010843:Pichia / Q000235:genetics*; D014176:Protein Biosynthesis; D011506:Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D014170:Transformation, Genetic
D015853:Cysteine Proteinase Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; C106356:equistatin