Fig. 1. IMAC/TAGZyme process development strategy. The E. coli-based process development starts with a screening for best expression clones and conditions followed by a scale-up of expression and purification and downstream processing of the target protein. Single-step purification using large-scale Ni–NTA Superflow chromatography is followed by desalting and TAGZyme tag processing and subtractive IMAC using the same Ni–NTA Superflow column. Eventual contaminations from the initial IMAC step rebind to the matrix in the subtractive step and are trapped along with the TAGZyme enzyme and unprocessed His-tagged protein resulting in a highly pure target protein preparation. The processes is checked by product quality control which may include 1D and 2D SDS–PAGE, SEC (size exclusion chromatography), DLS (dynamic light scattering), Western Blot, ELISA’s, ICP-MS (inductively coupled plasma-mass spectrometry), LAL (Limulus amoebocyte lysate), HCP (host cell protein assay), Threshold (host cell DNA assay), a protein activity assay, protein sequencing (Edman, MS/MS), and X-ray crystallography.

Fig. 2. IL-1β expression, purification, His-tag cleavage kinetics, and subtractive IMAC. Optimal expression conditions were: LB-media, 30 °C, induction with 1mM IPTG at OD_600nm of 1.0 M, marker (size indicated [kDa]); lane 1, cleared lysate; 2, flow-through fraction; 3, first wash fraction; 4, second wash fraction; 5–9, elution fractions 1–5; 10, His-tag IL-1β before addition of DAPase; 11–14, 30, 60, 90, 120 min after addition of DAPase; 15, IL-1β recovered after sIMAC.

Fig. 3. 2D gel electrophoresis analysis of His-tag IL-1β eluted from the initial Ni–NTA purification step (A) and of the DAPase-processed, detagged IL-1β (B).

Fig. 4. Quality control of IL-1β. (A and B) Immunologic analysis of DAPase removal. In the Ponceau-S stain (A), IL-1β is visible in lanes 2 and 3 at 17 kDa. (B) In the Western Blot, detection of DAPase heavy chain was by chemiluminescence using an anti-mouse HRP conjugate reporter. Lane 1, prestained marker (size indicated kDa); 2, aliquot of the cleavage reaction after 120 min containing 10 ng DAPase; 3, final IL-1β sample (0.625 μl). (C) ELISA analysis of Benzonase removal. Corresponding aliquots of each fraction were analysed. Detection limit of the assay for Benzonase is 0.2 ng/ml. CL, cleared cell lysate; FT, IMAC flow-through; W1 and 2, first and second IMAC wash; His-tag IL-1β, IMAC elution; IL-1β, sIMAC flow-through. (D) Chromatogram from size-exclusion chromatography of the final IL-1β sample. A Superdex 200 column was loaded with 3.5 mg (250 μl) of the processed protein and run with 1×TAGZyme buffer. The arrows indicate elution points of protein standards (from left to right: BSA, 66 kDa; GFP, 32 kDa; lysozyme, 14 kDa).

Fig. 5. 3D model of IL-1β from X-ray crystallography. Crystals in dimensions of 0.3 × 0.3 × 0.5 mm were obtained after 14 days in ammonium sulfate/Na-acetate solutions of acidic pH. The arrows indicate beta sheets and the lines in between coiled regions. The position of the N- and C-termini are indicated. (A) Whole molecule model. Three sulfate ions were determined and their exact positions in the vicinity of the N-terminus and their interactions are visualized. (B) Folding of and hydrogen bond-mediated intra- as well as intermolecular interactions at the N-terminus of IL-1β. Alanine 1 (ALA 1) of one IL-1β molecule interacts with glutamate 50 (GLU #50) of a neighboring molecule. See supplementary data on-line for crystal and diffraction pattern images.

Screening conditions for IL-1β expression

Recoveries of the IMAC/TAGZyme process steps

Sequences of IL-1β before and after His-tag cleavage

N-terminal amino acids representing the N-terminus of mature IL-1β is indicated in bold letters. N.d., not determined.

Quality control of IL-1β