SUMO fusion technology for difficult-to-express proteins - PubMed (original) (raw)

Review

SUMO fusion technology for difficult-to-express proteins

Tauseef R Butt et al. Protein Expr Purif. 2005 Sep.

Abstract

The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the field of protein production and purification. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good purification methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, purification, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems.

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Figures

Fig. 1

Fig. 1

The SUMO cycle. SUMO is synthesized as a precursor and cleaved by SUMO proteases (yeast Ulp1 or Ulp2). Activating enzyme (E1), conjugating enzyme (E2), and ligase (E3) are named according to yeast where most were discovered.

Fig. 2

Fig. 2

Enhanced expression and purification using the SUMO fusion system. (A) Enhanced expression of SARS-CoV 3CL protease (3CL) by SUMO fusion in E. coli. Cells grown in Luria–Bertani (LB) media were induced at the temperatures and for the lengths of time indicated. Just before expression was induced and after induction was completed the cells from a 1.5 ml aliquot of culture were lysed. Samples of whole cell lysates (∼7.5 μl) from the various expression conditions were resolved in 12% SDS-gels and stained with Coomassie blue. Molecular weights (in kDa) were as indicated, and arrowheads highlight expected/observed positions of respective expressed protein bands . (B) An SDS gel depicting the purification of SARS-CoV 3CL protease. Total cell lysate was passed over a Ni–NTA column, washed with 40 mM imidazole, and eluted with 300 mM imidazole (affinity purified). Cleavage with the SUMO protease was conducted under standard conditions, and the sample was passed over another Ni–NTA column to remove the SUMO protease and tag (subtracted). Aliquots of the samples (each containing ∼5 μg protein) were separated on a 12% SDS-gel and stained with Coomassie blue. The migration positions of the SUMO fusion and the proteins resulting from the cleavage are as indicated .

Fig. 3

Fig. 3

Effect of various conditions on the activity of SUMO protease. SUMO-green fluorescent protein fusion (SUMO-GFP) (2.5 μg) was incubated with SUMO protease (1:5000 molar ratio of SUMO-GFP to protease) for 1 h under conditions described in the figure: temperatures of 4 or 37 °C, and concentration ranges of imidazole (at 25 °C), sodium dodecyl sulfate (SDS) (at 25 °C), Triton X-100 (at 25 °C), urea (at 25 °C), or guanidine hydrochloride (at 25 °C). The data show that the enzyme is active over a broad temperature range and tolerates highly adverse biochemical conditions .

Fig. 4

Fig. 4

The X-ray crystal structure of human SUMO protease (Senp2, grey) in complex with human SUMO-1 (black) . SUMO-1 must pass through a constrictive hydrophobic tunnel (arrow) within the active site in order to be cleaved by Senp2.

Fig. 5

Fig. 5

The split SUMO expression system. (A) The structure of SUMO, and the N- and C-terminal halves (NTHS and CTHS). The target protein is fused to the CTHS, and the full SUMO structure is reconstituted after purification by incubating with NTHS. (B) The reconstitution of cleavable structure on the CTHS fusion in vitro and cleavage by Ulp1. An SDS PAGE of 6× His-CTHS-GFP (8 μg) fusion protein purified from E. coli that was incubated for 30 min at 30 °C with purified 6× His-NTHS (2 μg) and increasing concentrations of SUMO protease (lane 1, CTHS-GFP + NTHS and lanes 2–9, CTHS-GFP + NTHS with decreasing concentrations of SUMO protease (1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.8, and 3.9 ng)). Note the release of free GFP indicating the reconstitution of the full SUMO structure at high protease concentrations.

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