BACKGROUND:Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system.However,the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE:To obtain FLRT3 C-terminal gene fragments,to effectively express and purify the target proteins. DESIGN,TIME AND SETTING:An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine,Central South University between October 2007 and June 2008. MATERIALS:Three Sprague Dawley adult rats were used to extract total RNA from rat brains. The pGEX4T3 and Escherichia coli(E.coli)JM109 were purchased from Promega.E.coli BL21 was provided by Novagen. METHODS:FLRT3 protein coding C-terminal DNA fragments,at a length of 786 bp,were amplified using RT-PCR technique from rat total RNA.The amplified products were cloned into the expression vector pGEX4T3.A recombinant expression vector was then constructed and introduced into E.coli BL21.IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins,followed by isolation,purification,and renaturation of inclusion bodies that comprised recombinant proteins.Finally,the purified recombinant protein was obtained. MAIN OUTCOME MEASURES:Determination of FLRT3 C-terminal DNA sequence;expression of target proteins was assayed by SDS-PAGE electrophoresis;purified recombinant protein was identified with Western blot methods. RESULTS:FLRT3 protein coding C-terminal DNA fragments,at a length of 786 bp,were successfully harvested through RT-PCR amplification,and were then clones into the prokaryotic expression vector pGEX4T3.The results of the sequence were consistent with the known gene sequence.SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600.The recombinant protein was observed in the inclusion body,and highly purified recombinant proteins were obtained through a series of methods,such as rinsing,purifying,dissolving,and renaturing. CONCLUSION:From adult Sprague Dawley rats,FLRT3 C-terminal gene fragments were successfully cloned and shown to be effectively expressed in E.coli BL21.Moreover,highly purified GST fusion proteins were obtained. BACKGROUND: Studies have suggested that fibronectin leucine-rich transmembrane protein 3 (FLRT3) is related to injury and regeneration of the nervous system. Despite the expression and biological characteristics of these proteins remain poorly understood. OBJECTIVE: To obtain FLRT3 C-terminal gene DESIGN, TIME AND SETTING: An observational study of cellular and molecular biology was performed at the laboratory of Histology and Embryology in Xiangya School of Medicine, Central South University between October 2007 and June 2008. METHODS: FLRT3 protein coding C-terminal (FACS) DNA fragments, at a length of 786 bp, were amplified using RT-PCR technique from rat total RNA. The amplified products were cloned into the expression vector pGEX4T3.A r ecombinant expression vector was constructed and introduced into E. coli BL21.IsopropyI-D-thiogalactopyranoside was applied to induce expression of recombinant GST fusion proteins, followed by isolation, purification, and renaturation of inclusion bodies that became recombinant proteins. Finaally, the purified The recombinant protein was obtained. MAIN OUTCOME MEASURES: Determination of FLRT3 C-terminal DNA sequence; expression of target proteins was assayed by SDS-PAGE electrophoresis; purified recombinant protein was identified with Western blot methods. RESULTS: FLRT3 protein coding C-terminal DNA fragments , at a length of 786 bp, were successfully harvested through RT-PCR amplification, and were then clones into the prokaryotic expression vector pGEX4T3. The results of the sequence were consistent with the known gene sequence. SDS-PAGE analysis demonstrated that there was a specific protein band in the recombinant GST fusion proteins at a relative molecular mass of 56,600. recombinantin was observed in the inclusion body, and highly purified recombinant proteins were obtained through a series of methods, such as rinsing, purifying, dissolving, and renaturing. CONCLUSION: From adult Sprague Dawley rats, FLRT3 C-terminal gene fragments were successfully cloned and shown to be efficiently expressed in E. coli BL21.Moreover, highly purified GST fusion proteins were obtained.