Immobilization and characterization of peptide deformylase on surface of ampholytic ion modified hydrophilic magnetic beads

Abstract

Objective To compare the immobilization effect of 2 kinds of hydrophilic magnetic beads with Ni2+-nitrilotriacetic acid (Ni2+-NTA) group or carboxyl group and surface modified by ampholytic ions on Mycobacterium tuberculosis peptide deformylase (MtPDF), and to determine the target enzyme immobilization scheme appliable to screening MtPDF ligand mixture library using magnetic separation combined with high performance liquid chromatography-mass spectrometry (HPLC-MS). Methods The plasmids pET-28a(+): def with N-terminal 6×His tagged MtPDF was successfully constructed and transfected into E.coli BL21(DE3) for MtPDF recombinant expression. The products were collected and purified using nickel-ion chelating affinity chromatography column, and then identified with the enzymatic properties. Then the obtained MtPDF was coupled to magnetic beads with Ni2+-NTA (MtPDF-Ni2+-NTA) or carboxyl group (MtPDF-carboxy), respectively. The immobilization capacity, enzyme activity, enzyme stability before and after immobilization, and the response to the inhibitor actinonin were compared between the 2 schemes. Results The immobilization capacity of MtPDF-Ni2+-NTA was 99.7±4.5 mg/g (n=3). When the ratio of amount of enzyme to magnetic beads was 1 ∶15, the retention activity of the immobilized MtPDF was (107.2±13.9)% (n=3) of the free enzyme, and decreased to 43% after 3 h under shaking at 0 ℃ and pH value of 7.5. By contrast, the immobilization capacity of MtPDF-carboxy was 36.9±2.6 mg/g (n=3), and the retention activity of the immobilized MtPDF reached to (102.6±15.4)% (n=3) when the ratio of amount of enzyme to magnetic beads was 1 ∶60, consistent with that on the Ni2+-NTA magnetic beads, with negligible changes within 8 h under shaking at 0 ℃ and pH 7.5. The response of MtPDF-carboxy to the inhibition of actinonin had no significant difference from that of free MtPDF (P>0.05). Conclusion MtPDF-carboxy magnetic beads present stable retention activity, and has similar inhibitory response as that of free MtPDF, and thus is more suitable for screening of mixture library to discover high affinity ligands for MtPDF.

Keywords

• magnetic beads immobilization
• peptide deformylase
• tuberculosis
• screening