Molecular cloning of a cDNA clone for tobacco lipid transfer protein and expression of the functional protein in Escherichia coli.
A cDNA clone encoding a lipid transfer protein (LTP) was isolated from tobacco by screening a library with a PCR-amplified spinach LTP gene. DNA sequence analysis showed a large open reading frame (344 bp) encoding a polypeptide of 114 amino acids. The first 23 amino acids of the deduced protein have the characteristics of a signal peptide for protein secretion or targeting into dense microbody-like vesicles. The cDNA clone was then inserted into an expression vector, pMAL, and expressed in E. coli as a fusion with the maltose binding protein (MBP). The MBP-LTP fusion protein was purified to homogeneity and subjected to factor Xa cleavage to yield the LTP domain. A lipid transfer assay demonstrated that the resulting LTP was functional. The availability of the expression system in E. coli will facilitate the elucidation of in vivo function(s) of plant LTPs.
Masuta
C
C
Life Science Research Laboratory, Japan Tobacco Inc., Yokohama.
Furuno
M
M
Tanaka
H
H
Yamada
M
M
Koiwai
A
A
D000595:Amino Acid Sequence; D052179:Antigens, Plant; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D010940:Plant Proteins; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000235:genetics*
D052179:Antigens, Plant; D002352:Carrier Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C511920:lipid transfer proteins, plant; D004247:DNA