PCR

Yam chitinase E
Vacuolar targeting signal (VTS)

Yam chitinase E without VTS a-factor Yam chitinase E without VTS

AOX 1 AOX 1 TT
a-factor
ZeocinR
AOX 1 AOX 1 TT pPICZaA / YamE without VTS
Multiple cloning site

pPICZaA
ZeocinR
a-factor Yam chitinase E with VTS
Ligation
Yam chitinase E
AOX 1 AOX 1 TT

Vacuolar targeting signal (VTS)

ZeocinR
pPICZaA / YamE with VTS

Transformation of Eschericia coli JM109 and grown on LB medium containing ZeocinTM(at 37℃)

Large scale culture of positive colony in LB medium containing ZeocinTM(at 37℃)

Plasmid extraction

Linearization with restriction enzyme, PmeI at AOX 1 region

Transfection into Pichia pastoris X-33 strain by electroporation

Fig. 4. Outline of Yam class IV chitinase expression systems in P. pastoris. The vector
pBluescript SK+ and pDrive were used respectively for VTS and no-VTS yam chitinase cloning.