Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system.
Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.
Kang
Hye-Jin
HJ
Department of Biological Engineering, Inha University, Incheon 402-751, Korea.
Kim
Jin-Hee
JH
Chang
Woo-Jin
WJ
Kim
Eung-Soo
ES
Koo
Yoon-Mo
YM
D001483:Base Sequence; D004549:Elastin / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D006602:Hexosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D044367:Phase Transition; D011549:Pseudomonas / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D013696:Temperature
D011993:Recombinant Fusion Proteins; D004549:Elastin; D006602:Hexosyltransferases; C021513:levansucrase