An efficient system for high-level expression and easy purification of authentic recombinant proteins (1)
A fusion partner that has been used for some years is ubiquitin (Ub). It offers a natural yield enhancement, and, uniquely, the Ub moiety can be removed by highly specific proteases known as deubiquitylating enzymes (DUBs). This cleavage occurs precisely after the final glycine residue at the carboxyl terminal of Ub irrespective of the amino acid immediately following, with the sole exception of proline, which is cleaved inefficiently.
The pHUE vector was constructed for the expression of His-tagged ubiquitin fusion proteins by modifying pET15b. The ligated DNA fragment must encode Gly 75-Gly 76 residues of Ub, which are essential for cleavage.
Several pHUE constructs were made using a number of different genes. They in- cluded (1) SUMO, a small Ub-like protein; (2) human Pi class glutathione S-transferase, with an amino terminal me- thionine reside (M-GSTP1); (3) human Pi class glutathione S-transferase, with an amino terminal proline reside (P- GSTP1); (4) human glutathione synthetase (GSH-S); and (5) lacZ, encoding the E. coli protein-galactosidase (b-gal).
A substantial amount of protein was recovered despite a large amount lost in the flow-through by failing to bind to the Ni-NTA agarose beads. Only two proteins, Ub–bGSH-S and Ub-b-gal, failed to give a high recovery, which was due to their reduced solubility.
The catalytic core of the Usp2–45 open reading frame was cloned into pET15b for expression as a His-tagged protein. The expressed enzyme (termed Usp2-cc) was detectable as a predominant band on a Coomassie blue-stained SDS-PAGE gels. It was purified from crude E. coli extracts by nickel-affinity chromatography under native conditions at a final yield of ∼20 mg per liter of E. coli culture with ∼95% purity, as determined by densitometry.
Recovering almost all of the cleaved protein while successfully removing all other proteins present in the digest by Ni-NTA agarose purification. The N terminus of the cleaved and purified M-GSTP1 was sequenced by Edman degradation to confirm precise cleavage by Usp2-cc, returning a sequence identical to the expected amino acid sequence.
For GSTP1 as a fusion to Ub, the specific activity was found to be 82.0 ± 12.48 umole/min/mg, and for cleaved GSTP1 the specific activity was 88.1 ± 7.2 umole/ min/mg. Binding to the con- trol ovalbumin peptide or his-tagged Ub was far weaker. The Importin a/b heterodimer bound very tightly to the T-ag-NLS peptide with a Kd of 3.0 ± 1.0 nM, which is comparable to a Kd of 3.4 ± 0.1 nM for the synthetic T-ag NLS peptide.
