Heterologous expression,chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata  

作　　者：W.WP.Rodrigo R.S Dassanayake E.H.Karunanayake Y.I.N.Silva Gunawardene O.VDS.J.Weerasena 

机构地区：[1]Department of Chemistry,Faculty of Science,University of Colombo,90,Cumaratunga Munidasa Mawatha.Colombo 03,Sri Lanka [2]Institute of Biochemistry,Molecular Biology and Biotechnology,University of Colombo,90,Cumaratunga Munidasa Mawatha,Colombo 03,Sri Lanka [3]Molecular Medicine Unit,Faculty of Medicine,University of Kelaniya

出　　处：《Asian Pacific Journal of Tropical Medicine》2014年第2期85-92,共8页亚太热带医药杂志（英文版）

基　　金：supported by a grant(SIDA/2006/BT/04) awarded by National Science Foundation of Sri Lanka

摘　　要：Objective:To clone,express and purify a putative parasitic nematode specific protein of Setaria digitata(S.digitata),filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to he used for structural and functional analyses.Methods:To characterize uneharacterized gene of,S.digitata(SDUG),the herterologous expression of SDUG was carried out in the pET[cloned into pET45b(+)]expression system initially and co-expression of SDUC using chaperoiie plasmids pG-KJE8,pGro 7,pKJE7,pG-Tf2 and pTf16 containing chapcrone proteins of dnaK-dnaJ-grpE-groES-gro-E,groES-groEL,dnaK-dnaJ-grpE,groES-groEL-tig,and tig respectively,was carried out subsequently.Results:Expression of SDUG was seen when Escherichia coli strain BI.21(DE3)is used,while concentrating protein largely into the insoluble fraction.The co-expression of SDUG using chaperoiie plasmid mediated system indicated a significant increase of the protein in the soluble fraction.Of the chaperon plasniid sets,the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of2 mg/mL L-arabinosc and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28℃.Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography.SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of^24 kDa.Conclusions:SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.Objective:To clone,express and purify a putative parasitic nematode specific protein of Setaria digitata(S.digitata),filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to he used for structural and functional analyses.Methods:To characterize uneharacterized gene of,S.digitata(SDUG),the herterologous expression of SDUG was carried out in the pET[cloned into pET45b(+)]expression system initially and co-expression of SDUC using chaperoiie plasmids pG-KJE8,pGro 7,pKJE7,pG-Tf2 and pTf16 containing chapcrone proteins of dnaK-dnaJ-grpE-groES-gro-E,groES-groEL,dnaK-dnaJ-grpE,groES-groEL-tig,and tig respectively,was carried out subsequently.Results:Expression of SDUG was seen when Escherichia coli strain BI.21(DE3)is used,while concentrating protein largely into the insoluble fraction.The co-expression of SDUG using chaperoiie plasmid mediated system indicated a significant increase of the protein in the soluble fraction.Of the chaperon plasniid sets,the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of2 mg/mL L-arabinosc and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28℃.Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography.SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of^24 kDa.Conclusions:SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.

关 键 词：SETARIA digitata Uncharacterized gene CHAPERON CO-EXPRESSION 

分 类 号：S852.7[农业科学—基础兽医学]