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Cloning and expression of luffin-a gene from the seeds of Luffa cylindrical / 浙江大学学报·医学版
Journal of Zhejiang University. Medical sciences ; (6): 207-216, 2005.
Article in Chinese | WPRIM | ID: wpr-353216
ABSTRACT
<p><b>OBJECTIVE</b>To clone luffin-a cDNA from the seeds of Luffa cylindrical, and to obtain bioactive recombinant luffin-a protein using the expression vector pET-44a (+) in E. coli.</p><p><b>METHODS</b>The cDNA sequence encoding luffin-a was cloned from the fresh seeds of Luffa cylindrical by RT-PCR. The target DNA fragments were sequenced after T-A cloning. The luffin-a expression plasmid was constructed by inserting the luffin-a cDNA fragment into vector pET-44a (+). Luffin-a was expressed in E. coli by addition of IPTG into final concentration 1.0 mmol/L. The recombinant luffin-a was identified by SDS-PAGE. The biological activity of luffin-a protein was evaluated by using the MTT assay in HepG2 cells following fluid-phase endocytosis.</p><p><b>RESULTS</b>In comparison with the reported luffin-a, the homology of nucleotide sequence of the cloned luffin-a gene was 99.73%, while their amino acid sequences were identical. The solubility of recombinant protein was analyzed by SDS-PAGE, and the luffin-a was mainly produced in inclusion bodies. The recombinant luffin-a, renatured by dialysis of the denatured products, showed a similar cytotoxicity to ricin A chain.</p><p><b>CONCLUSION</b>The cDNA of luffin-a has been successfully cloned. The recombinant luffin-a protein expressed by E. coli is bioactive.</p>
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Plant Proteins / Seeds / Molecular Sequence Data / Base Sequence / Chemistry / Polymerase Chain Reaction / Amino Acid Sequence / Cloning, Molecular / Luffa / Ribosome Inactivating Proteins, Type 1 Language: Chinese Journal: Journal of Zhejiang University. Medical sciences Year: 2005 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Plant Proteins / Seeds / Molecular Sequence Data / Base Sequence / Chemistry / Polymerase Chain Reaction / Amino Acid Sequence / Cloning, Molecular / Luffa / Ribosome Inactivating Proteins, Type 1 Language: Chinese Journal: Journal of Zhejiang University. Medical sciences Year: 2005 Type: Article