[Cloning and expression of cDNA for maize nonspecific lipid transfer protein as well as calmodulin-binding activity analysis of the expression product].
Maize nonspecific lipid transfer protein (Zm-nsLTP) was cloned and expressed to investigate its CaM-binding activity. The cDNA of Zm-nsLTP was amplified using RT-PCR (Fig.1), and then inserted into the vector pET32a(+). The recombinant vector pET-Zm-nsLTP was expressed in E. coli BL21(DE3)trxB(-). Results of CaM-gel overlay assays (Fig.2) and CaM-sepharose pull-down experiments (Fig.3) indicated that recombinant Zm-nsLTP was bound to CaM in a Ca(2+)-independent manner, which is in accordance with the way that CaMBP-10 and Arabidopsis non-specific lipid transfer protein-1 (At-nsLTP1) are bound to CaM. The CaM-binding domain in Zm-nsLTP was mapped to the region of 47-60 amino acids (Fig.3), and online sequence analysis using Predict Protein program predicted that it has a BAA structure (Fig.4,5).
Bai
Wen-Yan
WY
Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.
Zhao
Li-Qing
LQ
Li
Zhen-Peng
ZP
Xie
Wan-Qin
WQ
Zhao
Yu-Long
YL
Li
Cui-Feng
CF
D000595:Amino Acid Sequence; D001483:Base Sequence; D002147:Calmodulin / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D003313:Zea mays / Q000235:genetics* / Q000378:metabolism
D002147:Calmodulin; D002352:Carrier Proteins; D018076:DNA, Complementary; D010940:Plant Proteins; C030363:lipid transfer protein