4.4. Protein Extraction and Purification

The number of purified protein batches for colicins M, K, U, and Ib was 9, 4, 8, and 13, respectively. In most cases, one batch was defined as an independently produced lot starting with the extraction of plant material and in some cases starting with a preparation of Agrobacterium cultures for infiltration of plant material.

For purification, plant material was extracted using the buffer containing 20 mM citric acid, 20 mM NaH₂PO₄, and 30 mM NaCl in a 5:1 (v/w) buffer:biomass ratio. The extraction was carried out at pH 4 in case of ColM and ColK and at pH 5.5 in case of ColIb and ColU. Ground leaf material supplemented with pre-chilled extraction buffer was incubated at room temperature under constant agitation for 30 min followed by centrifugation at 10,000× g for 15 min. The supernatant was filtered using Miracloth followed by incubation of the filtrate for 20 min at room temperature and centrifugation for 30 min at 10,000× g at 22 °C. The resulting supernatant was filtrated using filter discs with a pore size of 8–12 µm (TSP extract) and the final filtrate was used for further purification by cation exchange chromatography.

The filtrate was loaded on a CaptoMMC (GE Healthcare, Munich, Germany) column equilibrated with extraction buffer. The column was washed with extraction buffer to remove weakly bound non-target proteins. For further improvement of colicin purity, a second wash step was introduced using different amounts of elution buffer consisting of 50 mM Na₂HPO₄ (pH 7.84), 10 mM citric acid, and 1 M NaCl at a percentage adapted for each colicin as 10% for ColK and ColIb, 30% and 40% for ColM and ColU, respectively. The elution of ColM, ColK, and ColIb was carried out in a linear gradient until 100% of elution buffer concentration was reached over 6 column volumes. In the case of ColU, the elution was carried out in a step with 100% elution buffer. The eluted fractions were analyzed by SDS-PAGE and Coomassie staining using Instant Blue^TM staining solution (Expedeon, San Diego, CA, USA). The colicin containing fractions were pooled and dialyzed overnight at 4 °C against 20 mM Na₂HPO₄ (pH 7.5), 10 mM citric acid, and 50 mM NaCl. After dialysis, the proteins were frozen in liquid nitrogen and finally freeze-dried by lyophilization using freeze dryer Alpha 1–2 LDplus (Martin Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany) for long-term storage of the colicins.

Protein concentrations were determined using Pierce^TM BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). The colicin recovery upon purification was determined by SDS-PAGE analysis and Coomassie staining by comparison of different amounts of TSP extract and dialyzed purified target proteins to a known amount of BSA standard. The percentage of recombinant colicins of TSP in plant extracts and colicin samples after purification was calculated based on total protein concentration and concentration of recombinant colicin estimated by SDS-PAGE-based visual comparison to BSA.