Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris.
Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, 220 Han Dan Road, Shanghai 200433, China.
Hairong
H
Lu
Jinjiang
J
Huang
Guodong
G
Li
Kuikui
K
Ge
Hongyu
H
Wu
Qingshan
Q
Huang
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D015870:Gene Expression; D006801:Humans; D007610:Kallikreins / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D053491:Proteinase Inhibitory Proteins, Secretory / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000074162:Serine Peptidase Inhibitors, Kazal Type; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic; D014357:Trypsin / Q000378:metabolism
D053491:Proteinase Inhibitory Proteins, Secretory; D011994:Recombinant Proteins; C553928:SPINK6 protein, human; D000074162:Serine Peptidase Inhibitors, Kazal Type; C425336:KLK14 protein, human; D007610:Kallikreins; D014357:Trypsin