**5. Extraction and purification of SAF from** *E. coli*

300 Chromatography – The Most Versatile Method of Chemical Analysis

**2** containing standard protein markers.

2b

2c **Figure 2. (a)** Elution pattern of SIF from *S. aureus* after gel filtration through Sephadex G-100 column showing the presence of SIF in fractions 12-18 with a peak value in fraction 15. **(b)** Elution pattern of SIF from *S. aureus* obtained after DEAE cellulose column showing sperm immobilization activity in fractions 47-52 with peak value in fraction **49**. **(c)** SDS-PAGE of purified SIF, with **Lane1** containing DEAE cellulose purified and concentrated fraction. Molecular weight approximately, 20 kDa and **Lane** 

48 h old culture of *E. coli* grown in Luria broth was centrifuged at 10,000 g for 10 min at 4ºC. Cell free supernatant was prepared by passing the supernatant through a 0.22 µm Millipore filter. The cell pellet was washed twice with sterile PBS. Both the cell free supernatant and the washed cells were checked for spermagglutinating activity by incubating with semen samples. As the washed cells showed spermagglutinating activity, therefore further studies were carried out with washed cells. Extraction of sperm ligand from washed cells was done by salt treatment. The washed cells of *E. coli* (1000 ml, 48 h old cell culture) were incubated with 1, 2, 3, 4 and 5 M solution of NaCl under shake conditions (150 rpm) at 37ºC for different time intervals 2, 4, 8, 12 and 24 h, separately. The cells were centrifuged at 10,000 g for 30 min. The resulting cell pellet and supernatant (which was dialyzed against double distilled water overnight at 4ºC and passed through the UM05 Amicon filter) were analyzed for sperm agglutinating activity. As pellet did not show sperm agglutinating activity, further work was carried out with the supernatant. SAF could be efficiently extracted by treatment of cell pellet with 3 M NaCl for 12 h under shaking conditions (150 rpm) at 37ºC. Purification of crude sperm ligand consisted of filtration of dialyzed and concentrated fraction through a Sephadex G-200 column, equilibrated and eluted with PBS. Fractions of 3 ml each were collected and read at 280 nm on U.V. spectrophotometer. Fractions showing sperm agglutinating activity i.e. 6-9, with a peak in fraction 7 (Figure 3a) were pooled and concentrated using polyethylene glycol (PEG) 6000 at 4ºC. These fractions were applied to DEAE cellulose column. Final elution was done with 0.05, 0.1, 0.2, 0.4 and 0.6 M NaCl dissolved in PBS. Fractions of 4 ml each were collected and read at 280 nm on U.V. spectrophotometer. Most of the SAF could be eluted with PBS containing 0.4 M NaCl (fractions 46-49, Figure 3b). Fractions showing agglutinating activity were further pooled, dialyzed and concentrated. The protein content at each step was assayed by the method of Lowry et al. [19] against a standard curve calibrated with bovine serum albumin. PAGE of purified sperm ligand was carried out to check the purification status. Molecular weight estimation by SDS-PAGE, using the standard molecular weight markers showed the presence of ~71 kDa protein band (Figure 3c) [20].
