Purification and characterization of native and recombinant SaPIN2a, a plant sieve element-localized proteinase inhibitor.
SaPIN2a encodes a proteinase inhibitor in nightshade (Solanum americanum), which is specifically localized to the enucleate sieve elements. It has been proposed to play an important role in phloem development by regulating proteolysis in sieve elements. In this study, we purified and characterized native SaPIN2a from nightshade stems and recombinant SaPIN2a expressed in Escherichia coli. Purified native SaPIN2a was found as a charge isomer family of homodimers, and was weakly glycosylated. Native SaPIN2a significantly inhibited serine proteinases such as trypsin, chymotrypsin, and subtilisin, with the most potent inhibitory activity on subtilisin. It did not inhibit cysteine proteinase papain and aspartic proteinase cathepsin D. Recombinant SaPIN2a had a strong inhibitory effect on chymotrypsin, but its inhibitory activities toward trypsin and especially toward subtilisin were greatly reduced. In addition, native SaPIN2a can effectively inhibit midgut trypsin-like activities from Trichoplusia ni and Spodoptera litura larvae, suggesting a potential for the production of insect-resistant transgenic plants.
Wang
Zhen-Yu
ZY
State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
Ding
Ling-Wen
LW
Ge
Zhi-Juan
ZJ
Wang
Zhaoyu
Z
Wang
Fanghai
F
Li
Ning
N
Xu
Zeng-Fu
ZF
D000595:Amino Acid Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D052585:Phloem / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018547:Plant Stems / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D012697:Serine Endopeptidases / Q000378:metabolism; D032322:Solanum / Q000235:genetics / Q000378:metabolism*; D020860:Subtilisin / Q000037:antagonists & inhibitors / Q000378:metabolism; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D010940:Plant Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D020860:Subtilisin; D003546:Cysteine Endopeptidases