Enhanced production of recombinant HALT-1 pore-forming toxin using two-step chromatographic procedure

Hydra actinoporin-like toxin-1 (HALT-1) has been isolated from Hydra magnipapillata and is highly cytolytic against various human cells including erythrocyte. Previously, recombinant HALT-1 (rHALT-1) was expressed in Escherichia coli and purified by the nickel affinity chromatography. In this study, we improved the purification of rHALT-1 by two-step purifications. Bacterial cell lysate containing rHALT-1 was subjected to the sulphopropyl (SP) cation exchange chromatography with different buffers, pHs, and NaCl concentrations. The results indicated that both phosphate and acetate buffers facilitated the strong binding of rHALT-1 to SP resins, and the buffers containing 150 mM and 200 mM NaCl, respectively, removed protein impurities but retain most rHALT-1 in the column. When combining the nickel affinity chromatography and the SP cation exchange chromatography, the purity of rHALT-1 was highly enhanced. In subsequent cytotoxicity assays, 50% of cells could be lysed at [~]18 and [~]22 g/ml of rHALT-1 purified with phosphate and acetate buffers, respectively. O_LIHALT-1 is a soluble -pore-forming toxin of 18.38 kDa. C_LIO_LIrHALT-1 was purified by nickel affinity chromatography followed by SP cation exchange chromatography. C_LIO_LIThe cytotoxicity of purified rHALT-1 using 2-step purifications via either phosphate or acetate buffer was comparable to those previously reported. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/503828v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1b01c1borg.highwire.dtl.DTLVardef@1055147org.highwire.dtl.DTLVardef@1b2eb1org.highwire.dtl.DTLVardef@966fc0_HPS_FORMAT_FIGEXP M_FIG C_FIG Specifications table O_TBL View this table: org.highwire.dtl.DTLVardef@82c23forg.highwire.dtl.DTLVardef@9ede5dorg.highwire.dtl.DTLVardef@18e25aorg.highwire.dtl.DTLVardef@1b4b60dorg.highwire.dtl.DTLVardef@11c1eb7_HPS_FORMAT_FIGEXP M_TBL C_TBL