Example

TAGZyme Example

Purification of human tumor necrosis factor alfa (hTNFa) using the IMAC/TAGZyme strategy.

Human Tumour Necrosis Factor a (hTNFa) has an N-terminal sequence which does not constitute a natural DAPase stop point (Val-Arg-Ser-Ser-). The his-tag fusion protein Met-Lys(His-Gln)6-Gln-hTNFa (HT15-Gln-hTNFa) was expressed in E. coli and purified from a clarified crude extract by Ni(II)-IMAC.

Purified HT15-Gln-hTNFa (Figure, lane 2) was treated with DAPase (HT-DPP1) and Qcyclase at 37°C. A nearly quantitative conversion of HT15-Gln hTNFa (18.6 kDa) to a protein with a migration similar to that expected for pyroGlu-hTNFa (17.5 kDa) was observed after 30 minutes (Figure, lane 3-5). After completion of the reaction, the mixture was pumped through two serial connected columns, the first containing Ni(II)-Chelate-Sepharose FF and the second containing Zn(II)-Chelate-Sepharose FF charged with pGAPase. The protein flow-through from the passage of the two columns (Figure, lane 6) had the expected N-terminal sequence of hTNFa.

To further investigate the efficiency of the pGAPase catalyzed removal of pyroGlu residues, an aliquot of the fully processed TNFa was subjected to a second DAPase treatment (125 µg/mg TNFa, 2 hours) and SDS-PAGE. A quantitative DAPase catalyzed conversion of deprotected TNFa into truncated forms with increased electrophoretic mobility was observed confirming an efficient pGAPase catalyzed removal of the pyroGlu residue.

The yield of purified hTNFa from 20 mg HT15-Gln-hTNFa was 18 mg, corresponding to 90 %.