Purification of recombinant EGFP by fusion with L2 (252-273) from ribosomal protein L2 using magnetic particles.
A basic polypeptide L2 (252-273) derived from Escherichia coli ribosomal protein L2 was used as a purification tag. In order to develop faster, less expensive methods for expression and purification of proteins, the L2 (252-273)-small ubiquitin like modifier (SUMO) fusion expression system was constructed. We comparatively analyzed the adsorption properties of the deleted protein of L2 (L2 (252-273)) on diatomite and superparamagnetic carboxymethyl chitosan nanoparticles. The time required to reach adsorption equilibrium of L2 (252-273) fusion protein on diatomite was shorter than that of L2 (252-273) fusion protein on magnetic particles. The maximum adsorption capacity of L2 (252-273) fusion protein on magnetic particles was about 5 times larger than that of L2 (252-273) fusion protein on diatomite. SUMO was introduced as a specific protease cleavage site between the target protein and the purification tags. The enhanced green fluorescent protein (EGFP) as a model protein was fused with the L2 (252-273)-SUMO fusion protein and purified by a simple method which involves the electrostatic adsorption of L2 (252-273) fusion proteins on superparamagnetic carboxymethyl chitosan nanoparticles and the L2 (252-273)-SUMO fusion partner was removed based on the robust cleavage by the poly lysine tagged SUMO protease. The high purity of tag-free EGFP (>93%) was obtained. Our results preliminary proved that the system was an effective fusion expression system for the production of recombinant proteins in E. coli.
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Junhua
J
Li
Yiting
Y
Dong
Yang
Y
Zhang
Yanjun
Y
Yang
D000327:Adsorption; D000595:Amino Acid Sequence; D002412:Cations; D002852:Chromatography, Ion Exchange / Q000379:methods*; D007692:Diatomaceous Earth; D029968:Escherichia coli Proteins / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012269:Ribosomal Proteins / Q000737:chemistry*; D025842:SUMO-1 Protein / Q000737:chemistry; D055672:Static Electricity
D002412:Cations; D029968:Escherichia coli Proteins; D058185:Magnetite Nanoparticles; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D025842:SUMO-1 Protein; C485184:enhanced green fluorescent protein; C560088:ribosomal protein L2, E coli; D049452:Green Fluorescent Proteins; D007692:Diatomaceous Earth; C033787:diatomite