Figure 2. Expression of recombinant Colα3(IV)NC1 protein in bacteria, its purification, and its effects on the Sertoli cell TJ-permeability barrier function. (A) BL21 (DE3) competent E. coli cells transformed with pET46 Ek/LIC-Colα3(IV) NC1 vector was induced with 0.1 mM IPTG to express the recombinant protein. Strong expression of Colα3(IV) NC1 domain (28 kDa) was noted when compared the un-induced total cell protein (TCP) to induced TCP as shown in a representative Coomassie blue stained gel (A:a). Recombinant protein was purified by Ni²⁺-Sepharose chromatography in which the histidine (His, H) tag at the N-terminus of the recombinant protein specifically bound to the Ni-column. The eluted protein was purified to apparent homogeneity (A:b), and it was recognized by both anti-His (A:b) and anti-Colα3(IV) NC1 antibody (A:c) (see Table 1). (B) Primary Sertoli cells were plated at 1.2 × 10⁶ cells/cm² on Matrigel-coated bicameral units at time 0 to allow the assembly of a functional TJ-permeability barrier. Purified bacterial Colα3(IV) NC1 recombinant protein was refolded and dialyzed against PBS as described in Materials and Methods, and it was included in the F12/DMEM medium on day 2 at a concentration of 30 μg/ml (~1 µM). Mild but not statistically significant perturbation of the Sertoli cell BTB was observed when compared with PBS control (Ctrl) (Fig. 2B). Bacterial recombinant Colα3(IV) NC1 protein was removed 2 d after incubation. Each data point had n = 3 bicameral units. This experiment was repeated three times using different batches of Sertoli cells as well as recombinant protein, and yielded similar results.