Cloning and deletion
The full-length L1 clones of HPV16 and HPV18 in pGEX-2T were used as parent clones for generating the deletion mutants of D1, D2, D3, and D4 as shown in Fig. 2A, 2B. The L1 proteins were cloned downstream of GST to express as GST-L1 fusions in this E. coli expression vector. The standard molecular cloning methods, including PCR amplification, enzyme digestion, DNA end ligation, and transformation, was used to obtain the deletion mutants of L1 as shown in Fig. 2A, 2B. All deletion clones were confirmed by sequencing the whole DNA insert to ensure the correct deletion and the wilt type sequences for this study.
The protein expression and purification of all L1 deletion mutants was carried out essentially as described preciously [1,2]. Briefly, 0.2 mM IPTG was use to induce protein expression overnight at room temperature (RT). After cell lysis by sonication in buffer L (50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 1 mM DTT, 1 mM EDTA, 10 mM PMSF), urea (ultrapure grade) was slowly added to the lysate to a final concentration of 3.0 M. The mixture was incubated at RT for one hour with gentle shaking, and then dialyzed against three changes of buffer over an 18 hour period. After centrifugation at 25,000 × g for 75 min, the supernatant was passed through a glutathione affinity column to bind GST-L1 fusions. Usually, 10 ml glutathione resin was used for supernatant from 12 liter cell culture. The column was washed with 10× bed volumes of buffer L to wash away contaminating proteins. At this point, an additional washing step using 10× bed volumes of 2.3 M urea can clean up the small amount of the contaminated groEL. L1 was then cleaved from the GST fusion using thrombin (Sigma, T6634) in an approximate ratio of 100 μg GST-L1 to 1 NIH unit of enzyme. The digestion was carried out at 4°C overnight. L1 was further purified by Superdex-200 (60/16 column) size-exclusion chromatography. During the purification process, keep all the reagents at 4°C whenever possible.
Analysis of in vitro particle assembly by EM and size-exclusion chromatography
Wild-type L1 can assemble into particles under known in vitro assembly conditions [1,2]. The assembly reaction was performed by incubating purified L1 protein in assembly buffer (1 M NaCl, 40 mM sodium acetate, pH 5.4) at 25°C for 30 minutes, at a protein concentration of approximately 0.1 mg/mL. For EM analysis, samples were adsorbed to glow-discharged, carbon-coated copper grids (EM Science) and negatively stained with 2% uranyl acetate. Grids were examined in a JEOL 1200 transmission electron microscope at 80 kV and 60000× magnification. For size-exclusion chromatography analysis, samples were injected directly into the sample loop and onto a Superdex-200 column (16/60, Pharmacia) at a flow rate of 1 mL/minute in the assembly buffer. The protein peaks were detected by an UV monitor at a wavelength of 280 nM.