Purification of phage display-modified bacteriophage T4 by affinity chromatography

BMC Biotechnology

Table 1 Endotoxin content in bacteriophage preparations obtained in affinity chromatography of bacteriophages displaying affinity tags

Bacteriophage preparation	LPS activity (SD) [EU/ml]
Bacteriophage modified with GST tag and purified on glutathione Sepharose (Figure 3)	186 (124)
Bacteriophage modified with GST tag and purified on glutathione Sepharose, prolonged washing (Figure 4)	13 (2)
Bacteriophage modified with His tag and purified on Ni-NTA agarose (Figure 5)	253 (172)
Bacteriophage modified with His tag and purified on Ni-NTA agarose, prolonged washing (Figure 6)	16 (1)
Bacteriophage modified with GST tag and purified on Ni-NTA agarose (non-specific), prolonged washing	40 (2)
Bacteriophage modified with GST tag and purified on glutathione Sepharose, released by proteolysis	363 (47)
Bacteriophage modified with GST tag and purified on glutathione Sepharose, released by proteolysis, prolonged washing	11 (1)

The endotoxin assay was the Limulus Amebocyte Lysate Assay (Lonza, USA). It is based on activation of pro-enzyme in the Limulus Amebocyte Lysate by endotoxin of Gram-negative bacteria. The activated enzyme digests the non-colour substrate Ac-Ile-Glu-Ala-Arg-pNA, resulting in the molecule pNA that is spectrophotometrically detected at 405-410 nm after stopping the reaction with 25% acetic acid. The correlation between endotoxin concentration and absorbance is linear within the range 0.1-1 EU/ml; therefore all the samples were diluted to this range. All the probes were doubled for the detection. The concentration in a sample was estimated in comparison to the solutions of an endotoxin standard (supplied with the LAL kit), also doubled. Reaction conditions: 10 min. 37°C for the lysate, 8 min. 37°C for LAL substrate, then stopping the reaction and detection in 96-well plates. Only apyrogenic equipment was used (once and never reused) in the test.

ISSN: 1472-6750