RESUMO
Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E. coli. For the first time, the amplified cystatin coding region was expressed as a part of maltose-binding fusion protein using pMALc2X over-expression vector in TB1 strain of E. coli without affecting the recombinant bacterial growth. In comparison to the previously prepared recombinant SMC (rSMC), a high amount (~44 mg/L of bacterial cell culture) of purified fused SMC (fSMC) was obtained using single-step purification method. fSMC strongly inhibited papain activity in vitro as compared to Celosia single-domain cystatin. Purified fSMC may be used for basic biochemical, pharmacological or clinical studies without the cleavage of its fusion parts.
Assuntos
Sequência de Bases , Clonagem Molecular , Cistatinas/genética , Primers do DNA , Escherichia coli/genética , Helianthus/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genéticaAssuntos
Animais , Apoptose/genética , Cistatina B , Cistatinas/genética , Epilepsias Mioclônicas/genética , Humanos , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Epilepsias Mioclônicas Progressivas/genética , Proteínas do Tecido Nervoso/genética , Neurônios/patologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases não ReceptorasRESUMO
We have cloned a novel gene, Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library. Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found in the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the proteins of the CRES subfamily of the family 2 cystatins which are expressed specifically in the reproductive tract. CYMG1 protein shows 44% identity with mouse CRES and 30% identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 gene was specifically expressed in adult mouse testis. RT-PCR also showed that Cymg1 was expressed in testis and spermatogonial cells. Cymg1 expression level varied in the different developmental stages of mouse testis, and were coincidental with spermatogenesis and sex maturation. These results indicate that Cymg1 may play important roles in mouse spermatogenesis and sex maturation.