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 o...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 coil （E. coil） JM109 were purchased from Promega. E. coil 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. coil 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. coil BL21. Moreover, highly purified GST fusion proteins were obtained.展开更多
基金Supported by:the National Natural Science Foundation of China,No.30600224Supported by:the National Natural Science Foundation of China,No.30700438+2 种基金China's Post-doctoral Science Fund, No.20060390886Hunan Province Natural Science Foundation,No.06JJ30014 Hunan Province Scientific Program,No.2008FJ3138
文摘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 coil （E. coil） JM109 were purchased from Promega. E. coil 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. coil 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. coil BL21. Moreover, highly purified GST fusion proteins were obtained.
