Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris.
The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C-terminal amino acids of the precursor form on the inhibitor activity, the C-terminal precursor and the mature protein were both expressed. A third His-tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 40-160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine beta-trypsin compared to the precursor and His-tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.
Volpicella
M
M
Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Via Amendola 165/A, 70126, Bari, Italy.
Schipper
A
A
Jongsma
M A
MA
Spoto
N
N
Gallerani
R
R
Ceci
L R
LR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D005285:Fermentation; D007700:Kinetics; D008969:Molecular Sequence Data; D009149:Mustard Plant / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000254:growth & development; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D010946:Plants, Medicinal; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology
C405940:MTI2 protein, Sinapis alba; D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin