**4. Extraction and purification of sperm immobilization factor (SIF) from**  *S. aureus*

For extraction of SIF, *S. aureus* was grown in Brain Heart Infusion (BHI) broth for 72 h at 37°C under shaking conditions (150 rpm). The cell culture was subjected to centrifugation at

Isolation and Purification of Sperm Immobilizing/Agglutinating Factors from Bacteria and Their Corresponding Receptors from Human Spermatozoa 299

10,000 g for 30 min at 4°C. Both the resultant cell pellet and culture supernatant were checked for sperm immobilization activity. As the activity resided with cell supernatant it was subjected to ammonium sulphate precipitation so as to get 20, 40, 60, 80 and 100% saturation. SIF could be precipitated at 60-80% saturation. These precipitated proteins containing the bioactive molecule were dialyzed extensively against PBS and applied to Sephadex G-100 column (2 cm x 31 cm) equilibrated and eluted with PBS. The head pressure maintained to achieve a flow rate of 10 ml/h. Fractions of 3 ml each were collected, and the absorbance of each fraction was read at 280 nm. Fractions (12–18) showing the immobilization activity were pooled and concentrated against PEG 6000 at 4°C (Figure 2a). These fractions were then applied onto DEAE cellulose column. The column was equilibrated with PBS and head pressure was maintained to achieve a flow rate of 60 ml/h. 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 the absorbance was read at 280 nm. The fractions (47–52) that caused immobilization of spermatozoa were pooled and concentrated (Figure 2b). The fractions concentrated after DEAE cellulose chromatography were dialyzed, lyophilized, and subjected to PAGE analysis. The presence of a single band suggested apparent homogeneity of the protein. This purified fraction was then analyzed by SDS-PAGE against standard molecular weight markers (Figure 2c). The molecular weight of SIF was estimated

2a

to be ~20 kDa [18].

**Figure 1. (a)** Elution pattern of SAF from *S. aureus* after gel filtration through Sephadex G-200 column showing the presence of SAF in fractions 4-7 with a peak value in fraction 5. **(b)** Elution pattern of SAF obtained after DEAE cellulose column showing sperm agglutination activity in fractions 35-39 with peak value in fraction 37. **(c)** SDS-PAGE of purified SAF, with **Lane1** containing DEAE cellulose purified and concentrated fraction. Molecular weight approximately, 65 kDa and **Lane2** containing Standard protein markers.

10,000 g for 30 min at 4°C. Both the resultant cell pellet and culture supernatant were checked for sperm immobilization activity. As the activity resided with cell supernatant it was subjected to ammonium sulphate precipitation so as to get 20, 40, 60, 80 and 100% saturation. SIF could be precipitated at 60-80% saturation. These precipitated proteins containing the bioactive molecule were dialyzed extensively against PBS and applied to Sephadex G-100 column (2 cm x 31 cm) equilibrated and eluted with PBS. The head pressure maintained to achieve a flow rate of 10 ml/h. Fractions of 3 ml each were collected, and the absorbance of each fraction was read at 280 nm. Fractions (12–18) showing the immobilization activity were pooled and concentrated against PEG 6000 at 4°C (Figure 2a). These fractions were then applied onto DEAE cellulose column. The column was equilibrated with PBS and head pressure was maintained to achieve a flow rate of 60 ml/h. 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 the absorbance was read at 280 nm. The fractions (47–52) that caused immobilization of spermatozoa were pooled and concentrated (Figure 2b). The fractions concentrated after DEAE cellulose chromatography were dialyzed, lyophilized, and subjected to PAGE analysis. The presence of a single band suggested apparent homogeneity of the protein. This purified fraction was then analyzed by SDS-PAGE against standard molecular weight markers (Figure 2c). The molecular weight of SIF was estimated to be ~20 kDa [18].

298 Chromatography – The Most Versatile Method of Chemical Analysis

1a

1b

1c **Figure 1. (a)** Elution pattern of SAF from *S. aureus* after gel filtration through Sephadex G-200 column showing the presence of SAF in fractions 4-7 with a peak value in fraction 5. **(b)** Elution pattern of SAF obtained after DEAE cellulose column showing sperm agglutination activity in fractions 35-39 with peak value in fraction 37. **(c)** SDS-PAGE of purified SAF, with **Lane1** containing DEAE cellulose purified and concentrated fraction. Molecular weight approximately, 65 kDa and **Lane2** containing

Standard protein markers.

Isolation and Purification of Sperm Immobilizing/Agglutinating Factors from Bacteria and Their Corresponding Receptors from Human Spermatozoa 301

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

**6. Extraction and purification of SIF from** *E. coli* 

The isolate of *E. coli* showing immobilization of spermatozoa was grown in BHI broth under shaking conditions (150 rpm) at 37ºC for 72 h. The culture was centrifuged at 10,000 g for 15 min at 4ºC and cell-free supernatant was prepared by passing the supernatant through a 0.22 µm Millipore filter. The supernatant was then subjected to ammonium sulphate precipitation so as to get 20, 40, 60, 80, and 100% saturation. The precipitates so obtained were dissolved in a minimum amount of PBS. The precipitated protein was dialyzed against PBS under cold conditions and checked for sperm immobilization. SIF could be saturated at 60-80% of ammonium sulphate. Further purification of the factor was done by gel filtration through a Sephadex G-100 column (2 cm x 31 cm) equilibrated, and eluted with PBS.

3c) [20].

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

**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 2** containing standard protein markers.
