Characterization, cloning and expression of the ferritin gene from the Korean polychaete, Periserrula leucophryna.
Ferritin is a major eukaryotic protein and in humans is the protein of iron storage. A partial gene fragment of ferritin (255 bp) taken from the total RNA of Periserrula leucophryna, was amplified by RT-PCR using oligonucleotide primers designed from the conserved metal binding domain of eukaryotic ferritin and confirmed by DNA sequencing. Using the 32P-labeled partial ferritin cDNA fragment, 28 different clones were obtained by the screening of the P. leucophryna cDNA library prepared in the Uni-ZAP XR vector, sequenced and characterized. The longest clone was named the PLF (Periserrula leucophryna ferritin) gene and the nucleotide and amino acid sequences of this novel gene were deposited in the GenBank databases with accession numbers DQ207752 and ABA55730, respectively. The entire cDNA of PLF clone was 1109 bp (CDS: 129-653), including a coding nucleotide sequence of 525 bp, a 5'-untranslated region of 128 bp, and a 3'-noncoding region of 456 bp. The 5'-UTR contains a putative iron responsive element (IRE) sequence. Ferritin has an open reading frame encoding a polypeptide of 174 amino acids including a hydrophobic signal peptide of 17 amino acids. The predicted molecular weights of the immature and mature ferritin were calculated to be 20.3 kDa and 18.2 kDa, respectively. The region encoding the mature ferritin was subcloned into the pT7-7 expression vector after PCR amplification using the designed primers and included the initiation and termination codons; the recombinant clones were expressed in E. coli BL21(DE3) or E. coli BL21(DE3)pLysE. SDS-PAGE and western blot analysis showed that a ferritin of approximately 18 kDa (mature form) was produced and that by iron staining in native PAGE, it is likely that the recombinant ferritin is correctly folded and assembled into a homopolymer composed of a single subunit.
Department of Bioindustry, College of Life & Environment, Daegu University, Kyungbuk 712-714, Republic of Korea.
Byeong Ryong
BR
Jeong
Su-Mi
SM
Chung
Nam Joo
NJ
Baek
Kwang Bon
KB
Koo
Hyung Suk
HS
Baik
Han-Seung
HS
Joo
Chung-Soon
CS
Chang
Jang Won
JW
Choi
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005293:Ferritins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D035925:Iron-Regulatory Proteins / Q000737:chemistry / Q000378:metabolism; D007723:Korea; D051379:Mice; D008969:Molecular Sequence Data; D011077:Polychaeta / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D035925:Iron-Regulatory Proteins; D005293:Ferritins