Expression of HPV 6b L1 protein and its short variant in silkworm larvae
HPV 6b L1 protein has a basic domain at its C-terminus, which can bind to genomic DNA and pack into virus particles (Li et al. [1997]; Touze et al. [2000]). Full-length L1 and short proteins lacking its basic domain at its C-terminus (Figure 1B) were expressed in silkworm larvae. Both the L1 protein and its variant were expressed in the fat body of silkworm larvae, but not in hemolymph (data not shown). In sucrose density gradient centrifugation analysis, many bands that derived from L1 full-length protein were detected mainly in fraction 1, but these bands have lower molecular weight than that of full-length L1 (Figure 2A). These low-molecular weight L1 proteins may come from proteolytic degradation by some proteases in silkworms. Full-length L1 protein was detected mainly in between fractions 3 and 5. On the other hand, short L1 protein was observed mainly in between fractions 1 and 4 (Figure 2B). Full-length L1 protein was observed in a slightly higher sucrose density fraction compared to short L1 protein, suggesting that full-length L1 protein may form large particles.
After the partial purification of full-length and short L1 proteins from the fat body of silkworm larvae by heparin affinity column chromatography, both purified full-length and short L1 proteins were analyzed by SDS-PAGE, western blot, and electron microscope. Minor low-molecular weight bands were detected by SDS-PAGE (Figure 3I-A and 3II-A), and their molecular weights were also confirmed by western blot (Figure 3I-B and 3II-B). Full-length L1 proteins formed heterogenous VLPs, which have 10–50 nm in diameter (Figure 3I-C). HPV-VLPs have a diameter of 55 nm, suggesting that large VLPs have a normal size of HPV-VLPs in a T=7 icosahedral lattice, which consists of 72 pentamers, and small VLPs are T=1 particles, which consist of 12 pentamers or capsomeres. On the other hand, short L1 protein formed only small particles (Figure 3II-C). This result suggests that C-terminal basic domain in L1 protein may be required to form T=7 particles. Touzé et al. previously reported that Δ31 L1 protein from HPV 16, where 31 amino acids at its C-terminus were deleted, were still able to form T=7 particles, but deletion of 103 amino acids at its C-terminus caused the disruption of L1-VLP formation (Touze et al. [2000]). Moreover, the deletion of 22 amino acids in HPV 16 L1 protein did not have any effect on the assembly of T=7 VLPs. Most of the C-terminal-deleted L1 variant formed T=7 VLPs (Varsani et al. [2006]). Bian et al. reported that HPV 16 L1 ΔC34 (deleted 34 amino acids at its C-terminus) fused with an N-terminal domain (1–70 amino acids) of HPV E7 formed 11–12 nm of capsomeres, but not as large as VLPs (Bian et al. [2008]). These previous reports were obtained from the Escherichia coli expression system, but in this study, bacmid-silkworm expression system was used. These results suggest that protein expression system may have some influences on the properties of expressed L1 proteins.
Expression of L1 protein inserted with EGFP at its loop domain
To date, an antigen display system, found on the surface of VLPs targeting for efficient vaccines to infectious diseases, has been established using chemical conjugation, gene manipulation for the identification of immunogenic domains, conformation, and neutralizing epitopes of capsid proteins. Crystal structures of HPV 11, 16, 18, and 35 L1 proteins have been already solved, and L1 proteins have 5 loop regions (BC, DE, EF, FG, and HI loops) (Bishop et al. [2007]). Chimeric HPV 16 L1 proteins were constructed by the introduction of six amino acids coding hepatitis B core (HBc) antigen epitope (DPASRE) into 5 loop regions in HPV 16 L1 protein (Sadeyen et al. [2003]). These chimera L1 protein-formed T=7 particles and induced anti-HBc antibodies indicate that HBc epitope can be displayed on HPV VLP’s surface by its insertion into the loop region of L1 protein. Moreover, several heterogenous epitopes were also displayed on its surface by chemical conjugation and genetic manipulation (Murata et al. [2009]; Pejawar-Gaddy et al. [2010]; Schellenbacher et al. [2009]). In this study, whole protein was inserted into BC, EF, and HI loops of HPV 6b L1 protein by genetic manipulation. The alignment search was performed between the amino acid sequences of HPV 16 L1 and 6b L1 proteins, and putative each loop region in HPV 6b was described in Figure 1A. Each fusion protein (L1-55-EGFP, L1-174-EGFP, and L1-348-EGFP) was constructed as Figure 1B, and expressed in silkworm larvae. Three fusion proteins were detected by anti-L1 antibody and anti-EGFP antibody in the fat body at the estimated molecular weight, but most of the expressed protein was degraded (data not shown). These three fusion proteins were partially purified by heparin affinity column chromatography using almost the same protocol as the purification of full-length and short HPV L1 proteins, and were analyzed by electron microscope (Figure 4A). Approximately 10–20 nm of particles, capsomeres, and amorphous aggregates were observed in all samples, but VLP particles were not. By immunoelectron microscopy using an anti-EGFP antibody as the primary antibody, gold particles were observed on the capsomeres and aggregates in all samples (Figure 4B). EGFP fluorescence of partially purified L1-EGFP fusion protein was observed, but all L1-EGFP fusion protein did not have any fluorescence. These results indicate that L1-EGFP fusion proteins form capsomeres, and EGFP can be displayed on the surface of L1 capsomere without native EGFP conformation. In a previous report, 95 amino acids (HPV 16 L2 13–107 amino acids) were genetically inserted into the DE loop in bovine papillomavirus (BPV), and this L1 fusion protein did not form unequivocal VLPs but formed capsomeres and its aggregates. However, this fusion protein induced a slight but significant antibody response to HPV L2 protein when this L1-L2 fusion protein was immunized to rabbits with a native form (Schellenbacher et al. [2009]). These results suggest that it is possible that the insertion of long polypeptide (100 a.a.~) into the loop domain in HPV L1 protein disturbs VLP formation, but L1 protein inserted with a long polypeptide forms capsomeres and its L1-long polypeptide fusion protein capsomeres are prone to aggregate. In this study, three fusion proteins formed aggregates.