Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli.
The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1alpha (stromal cell-derived factor-1alpha) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1alpha fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP-RANTES and MBP-SDF-1alpha was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1alpha.
Cho
Hee-Jeong
HJ
School of Life Sciences and Biotechnology, Korea University, Anam-dong, Seungbuk-gu, Seoul, South Korea.
Lee
Young
Y
Chang
Rae Sung
RS
Hahm
Moon-Sun
MS
Kim
Myung-Kuk
MK
Kim
Young Bong
YB
Oh
Yu-Kyoung
YK
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D018946:Chemokine CCL5 / Q000302:isolation & purification / Q000378:metabolism*; D054377:Chemokine CXCL12 / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism*; D006801:Humans; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*
D002352:Carrier Proteins; D018946:Chemokine CCL5; D054377:Chemokine CXCL12; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase