Cloning and characterization of a cDNA for manganese superoxide dismutase from callus of sweet potato.
Lin C.T. | Lin M.T. | Shaw J.F.
A full-length complementary DNA (cDNA) clone encoding a putative manganese superoxide dismutase (Mn-SOD) from the callus of sweet potato was amplified by polymerase chain reaction technique from cDNAs synthesized from callus mRNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprises a complete open reading frame coding for 233 amino acid residues and a 29 amino acid transit peptide at the N terminus. The deduced amino acid sequence showed greater identity (67-75%) with plant mitochondrial Mn-SOD than with Mn-SOD from mammalian mitochondria (53%), yeast (47%), or bacteria (44%). The residues required to coordinate the single tervalent manganese ion and the 11 residues putatively involved in the active center are conserved as they are among all reported Mn-SOD sequences. These suggest that the sweet potato callus cDNA clone encodes a mitochondrial Mn-SOD. In addition, the coding region of Mn-SOD cDNA from sweet potato callus was introduced into an expression vector, pET-28a(+) and transformed into Escherichia coli BL21(DE3). A predominant protein band was detected by Coomassie blue staining of native PAGE, and activity staining confirmed the result of Coomassie blue staining. These indicate that this Mn-SOD cDNA clone can express Mn-SOD enzyme in E. coli.
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