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). This small eukaryotic protein provides two benefits. The Ub moiety can be removed by highly specific proteases known as deubiquitylating enzymes (DUBs) that do not cleave non- specific sequences and do not leave additional amino acids at the N terminus of the protein of interest.
The pHUE vector was constructed for the expression of His-tagged ubiquitin fusion proteins by modifying pET15b. It contains the inducible T7 RNA polymerase promoter, a histidine tag at the 5 end of a Ub open reading frame and an extended polylinker. When the presence of extra residues at the N terminus of the protein cannot be tolerated, a precise fusion to Ub can be generated using the SacII site, which has been engineered into the 3 end of Ub.
Each Ub fusion protein was detected as an abundant band on Coomassie blue-stained SDS gels, reflecting a high level of protein expression regardless of protein size.
Each of the His-tagged Ub fusion proteins was successfully purified from crude E. coli extracts by nickel-affinity chromatography under native conditions.
The expressed enzyme (termed Usp2-cc) was detectable as a predominant band on a Coomassie blue-stained SDS-PAGE gels. Various Ub time-course cleavage assays were performed and quantified by densitometry to optimize Usp2-cc activity.
Recovering almost all of the cleaved protein while successfully removing all other proteins present in the digest. 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.
The Importin alpha/beta 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 and to published values of 3.0 nM. Correction of the values from Figure 5A by subtracting the His6-Ub control gave a better curve fit (correlation coefficient of 0.989 versus 0.93) and a Kd of 1.7 ± 0.2 nM.
