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 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 $^{32}P-labeled$ 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.
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 $^{32}P-labeled$ 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.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
An indirect test of the biological function of the recombinant ferritin was conducted. Subunit assembly and core formation are assumed to represent necessary events in the path to biologic function, therefore, a modified Perl's staining (Chung, 1985; Seo et al.
The titer of the amplified cDNA library was determined to be 5 x 10r° pfu/ml. Using the ExAssist helper phage and the E. coli SOLR strain, an efficient excision of the pBluescript phagemid from the Uni-ZAP XR vector was carried out to yield various sizes of the phagemid. When the phagemids were digested with EcoRI and Xhol, insert DNAs ranging from 0.
대상 데이터
Polychaetes (Periserrula leucophryna) were captured in the tidal flats of Kwanghwa Island in the Korean west sea. Total RNAs were isolated from various tissues using TRIzol reagent (Sigma, USA) and QIAGEN RNA extraction kit or by the guanidinium/ phenol method (Chomczynski and Sacchi, 1987).
성능/효과
Partial cDNA products were obtained from total RNA or total cDNA of Periserrula leucophryna. As a result of the sequencing and BLAST search, one kind of novel ferritin cDNA fragment was obtained and the exact size of the partial gene was determined to be 255 bp in length. This clone was named the No.
By using 5 gg of poly(A)+RNA as a template; a cDNA library was synthesized according to the instruction manual as described by the manufacturer (Stratagene, USA). As a result, the efficiency of the cDNA library was calculated to be 8 x io5 plaque forming units (pfu)/gg cDNA containing approximately 97% recombinant phages. Because a primary library can be unstable, the library was immediately amplified in one round in the E.
In conclusion, we have cloned and characterized the novel ferritin gene of P. leucophryna and subsequently have produced the ferritin protein in E. coli by using the T7 promoter/T7 RNA polymerase system. It is difficult to speculate on the physiologically special properties of ferritin in P.
11 clone. It was confirmed that the DNA sequence incorporating the PCR primers faithfully reflected the actual DNA sequence of the amplified gene fragment. By using the ClustalV multiple sequence alignment programs (Higgins and Sharp, 1989), multiple alignments of the translated sequence of the ferritin PCR fragment, with their best match candidate, indicated a striking conservation of a number of residues and metal binding sites within the sequences.
후속연구
Characterization of the over-expressed gene products will provide additional biochemical information on the function of this enzyme protein and the role played in the physiology of this organism. Further studies will be designed to improve the expression efficiency, enhance protein production and purification and focus on the biological properties of the ferritin. Moreover, studies on the bioavailability of ferritin will be followed.
참고문헌 (34)
Andrew, S.C., P. Arosio, W. Bottke, J.F. Briat, M. von Darl, P.M. Harrison, J.P. Laulhe`re, S. Levi, S. Lobreaux, and S.J. Yewdall. 1992. Structure, function, and evolution of ferritins. J. Inorg. Biochem. 47, 161-174
Beck, G., T.W. Ellis, G.S. Habicht, S.F. Schluter, and J.J. Marchalonis. 2002. Evolution of the acute phase response: iron release by echinoderm (Asterias forbesi) coelomocytes, and cloning of an echinoderm ferritin molecule. Dev. Comp. Immunol. 26, 11-26
Brands, S.J. (comp.) 1989-2002. Systema Naturae 2000. Amsterdam, The Netherlands. [http://sn2000.taxonomy.nl/]
Chomczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156-159
Chung, M.C. 1985. A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferring and lactoferrin. Anal. Biochem. 148, 498-502
Gourley, B.L., S.B. Parker, B.J. Jones, K.B. Zumbrennen, and E.A. Leibold. 2003. Cytosolic aconitase and ferritin are regulated by iron in Caenorhabditis elegans. J. Biol. Chem. 278, 3227-3234
Didsbury, J.R., E.C. Theil, R.E. Kaufman, and L.F. Dickey. 1986. Multiple red cell ferritin mRNAs, which code for an abundant protein in the embryonic cell type, analyzed by cDNA sequence and by primer extension of the 5'-untranslated regions. J. Biol. Chem. 261, 949-955
Ha, Y., D. Shi, W. Small, E.C. Theil, and N.M. Allewell. 1998. Accession No. GI 5542291, unpublished
Harrison, P.M., P.J. Artymiuk, G.C. Ford, D.M. Lawson, J.M.A. Smith, A. Treffry, and J.L. White. 1989. p. 257-294. In Biomineralization: Chemical and Biochemical Perspectives(Mann, S., Webb, J. and Williams, R. P. J., eds.). VCH, Weinheim
Harrison, P.M. and P. Arosio. 1996. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1275, 161-203
Hempstead, P.D., S.J. Yewdall, A.R. Fernie, D.M. Lawson, P.J. Artymiuk, D.W. Rice, G.C. Ford, and P.M. Harrison. 1997. Comparison of the three-dimensional structures of recombinant human H and horse L ferritins at high resolution. J. Mol. Biol. 268, 424-448
Kim, H.G., T.N. Phan, T.S. Jang, M.J. Koh, and S.W. Kim. 2005. Characterization of Methylophaga sp. Strain SK1 cytochrome cL expressed in Escherichia coli. J. Microbiol. 43, 499-502
Kim, Y.M., K. Park, S.H. Jung, J.H. Choi, W.C. Kim, G.J. Joo, and I.G. Rhee. 2004. Chlorothalonil-biotransformation by glutathione s-transferase of Escherichia coli. J. Microbiol. 42, 42-46
Lacks, S. and B. Greenberg. 1977. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114, 225-232
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685
Le, N.T.V. and D.E. Richarson. 2002. The role of iron in cell cycle progression and the proliferation of neoplastic cells. Biochim. Biophys. Acta 1603, 31-46
Orino, K., K. Eguchi, T. Nakayama, S. Yamamoto, and K. Watanabe. 1997. Sequencing of cDNA clones that encode bovine ferritin H and L chains. Comp. Biochem. Physiol. 118B, 667-673
Orino, K., L. Lehman, Y. Tsuji, H. Ayaki, S.V. Torti, and F.M. Torti. 2001. Ferritin and the response to oxidative stress. Biochem. J. 357, 241-247
Park, R.Y., H.Y. Sun, M.H. Choi, Y.H. Bai, and S.H. Shin. 2005. Staphylococcus aureus Siderophore-Mediated Iron- Acquisition System Plays a Dominant and Essential Role in the Utilization of Transferrin-Bound Iron. J. Microbiol. 43,183-190
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. In Molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press
Seo, H.Y., Y.J. Chung, S.J. Kim, C.U. Park, and K.S. Kim. 2003. Enhanced expression and functional characterization of the human ferritin H- and L-chain genes in Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 63, 57-63
Studier, F.W., A.H. Rosenberg, J.J. Dune, and J.W. Dubendorff. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60-89
Tabor, S. and C.C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82, 1074-1078
Theil, E.C. 1987. Ferritin: structure, gene, regulation, and cellular function in animals, plants, and microorganisms. Ann. Rev. Biochem. 56, 289-315
von Darl, M., P.M. Harrison, and W. Bottke. 1994. cDNA cloning and deduced amino acid sequence of two ferritins: soma ferritin and yolk ferritin, from the snail Lymnaea stagnalis. Eur. J. Biochem. 222, 353-366
Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 114, 81-83
Zhang, Y., Q. Meng, T. Jiang, H. Wang, L. Xie, and R. Zhang. 2003. A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata). Comp. Biochem. Physiol., Part B Biochem. Mol. Biol. 135, 43-54
※ AI-Helper는 부적절한 답변을 할 수 있습니다.