Several strategic steps had already been reported in overcoming HPV cancers, and also, diverse difficulties need to be solved for a successful L1 protein expression, in order to meet the protein demands. The expression of L1 in E. coli was reportedly low, forming inclusion bodies that induce misfolded protein. Evidences suggest the truncation of the N-terminal and exclude the strong secondary structure inhibitor elements [17]. A recombinant construct of L1-HPV52 protein with 26 aa deletion of the N-terminal was developed, using an advanced pETSUMO expression system. Cloning of L1-HPV52 into appropriate vector is crucial for more efficient protein production that generates a high yield. The pETSUMO expression system employs TA cloning method that assures fast and efficient function [19]. Despite the challenges of using a bacterial expression system that generates inclusion bodies, pETSUMO solves the problem by enhancing the solubility of the partially insoluble protein. The SUMO tag became covalently conjugated to other proteins via an amide linkage, between C-terminal carboxyl and amino group in a lysine side chain [20]. Additionally, the expression system enabled the production of native protein by eliminating the poly histidine-tag SUMO fusion protein, which in turn, potentially affects the native conformation of the target protein [21].
This study showed that the recombinant protein with fusion tag remained in the resin when elution was done by 250 mM imidazole. It was hypothesized that poly histidine tag promotes oligomerization of the recombinant protein, which indicates higher imidazole concentration is needed to elute the protein [7]. Since the washing process is composed a low imidazole, the steps should be optimized to maintain the target protein. On the other hand, buffers play a significant role to maintain protein stability, where most of the protein stability was corroborated above the isoelectric point (pI). The isoelectric point of His-SUMO-L1HPV52 protein was 6.8, and the elution buffer used during the purification step was pH 8.0. The stability of the purified protein in this buffer did not persist for a long time because of the protein degradation process; therefore, the antibody was unable to detect any trace of purified protein through western blot analysis (data not shown). The occurred degradation process was caused by high concentrations of imidazole component in the elution buffer. The role of imidazole in protein degradation was explained as a catalytic reaction on histidine residues, therefore, purified protein with His-tag fusion was recommended to undergo dialysis with a buffer that maintains protein stability during storage [14].
In addition, some proteins with His-tag fusion are least stable in solutions for having pH values close to or lower than the calculated amount [22]. To overcome this challenge, the cleaved recombinant was directly processed further while it was in the resin, without eluting the protein and this decision successfully generated a soluble protein with single band at 55 kDa, which then was considered as an optimal procedure, to obtain the highest yield and concentration. Furthermore, the cleaved product was also comparable with the commercial L1 that was confirmed by immunoblot analysis. The SUMO fusion prevents protein aggregation even after the cleavage, due to its chaperone-like function to assist proper folding. Growth condition at 30 °C is good for expression of many SUMO fusion proteins, however, whenever the protein target is found insoluble at this temperature, it is then necessary to explore lower degrees (down to 15°C) [15]. In correlation to those previous findings, our results show similar outcomes, where the expression was performed at 20°C, it generated good results before and after the fusion cleavage.
In addition, to maintain the soluble native protein after cleavage and assembly into VLP, the L1-HPV52 was incubated in the low pH condition. The pH plays an important role for in vitro assembly, since it affects capsid-protein charge. Additionally, low temperatures are normally favorable as they reduce protein aggregation and chemical degradation. The L1 VLPs composed of 72-pentamers. Former research on HPV L1 has found that deletion of ten N-terminal residues led to assembly of a 12-pentamers rather than 72 [17]. Another research discoveries on Norovirus-like particles state that deletion of 34 and 98 amino acids of GII.4 Sydney (VP1) VLPs did not show any detectable particle with electron microscopy, however deletion of 26 and 38 amino acids introduced VLPs assembly [18]. This study is more likely to support the previous finding, where 26 amino acids truncated L1 protein successfully assembled into VLPs.
It is known that the size and homogeneity of observed particles depend on N-terminal truncation [15]. In our study, purified L1 VLP HPV52 showed variable particle size with mean ~26 nm, while the final yield of VLPs obtained was ± 6 mg/L. The heterogeneous sizes of the HPV L1 VLPs among different types were caused by a varied amino acid sequence in the N-terminal domains. Evidence suggests that the first 129 nucleotides in the 5′-end are composed of a strong RNA inhibitory component, and at least 10 and 30 residues were deleted from the N and C-terminus [17]. Truncation of ten residues in the N-terminal generated small L1 11/16 VLP with ~30 nm diameter [23], while 15 amino acid truncation generated L152 VLP with ~ 55 nm diameter [24].
The bioinformatic study out of sequencing result for L1HPV52 gene that has been inserted into pETSUMO has a total length of 1476 bp with 2 stop codons (TAA and TGA) on downstream of the gene. It expresses the major capsid protein (L1) HPV52 with a sequence length of 490 aa. Prediction of molecular weight, using bioedit v7.2, is 54846.63 Daltons (Da) or 54.9 kDa. The size is smaller than the native L1HPV52 because as many as 117 bp (39 aa) in the upstream of the gene were removed. The purpose of this partial deletion is based on a research conducted by Wei M and colleagues in 2018 [24], where they found that removal of 15 aa in the N-terminal of L1 HPV52 can increase their soluble expression in E. coli and in vitro self-assembly.
The B cells have an important role in HPV-associated cancer immunotherapy and response to cervical epithelial neoplasms and invasive cancers caused by HPV [26]. The EIDB results show that there are 17 B cell epitopes of L1HPV52. The sizes are varied, from 1 until 25 aa. Among those 17 candidates, 5 epitopes were selected, namely numbers 1, 2, 10, 12, and 16, based on their aa lengths that are neither too long nor too short. Other related studies of B cell epitope mentions that selection of a specific aa is usually made if it has not too long and short sequences, such as epitope studies on HPV 16 [27] and HPV33 and 58 [28]. In addition, this epitope has no mutations (conserved region) when it was aligned to 80 full coding sequences of L1HPV52 sin NCBI Genbank (Data not shown).
From these 5 epitopes, the 3 of them are located on the outer side of L1HPV52 protein, in all forms of L1HPV52 (monomer, pentamer, and VLP). These outer B cell epitopes were chosen regarding the fact that B cell only can recognize the outer epitope of an antigen. Attracting the B cells is important due to its function during the phagocytosis process when there is an antigen enters the body. If these three epitopes (number 3, 10, and 16) are recognized by B cell, then this B cell will engulf and degrade/break L1HPV52 antigen into smaller parts of peptides. Antigen phagocytosis by B cells is required for a potent humoral response [29].
The T cell epitope is associated with human leukocyte antigen (HLA). The HLA class I regions (HLA-A, B, and C) are carrying the highly polymorphic gene and those unique characteristic makes HLA precisely fit within its interaction through immunology view. The HLA class II regions (DP, DN, DM, DO, DQ, DR) are involved in antigen processing and presentation. While, the class III regions, contain genes that are implicated in inflammatory responses, leucocyte maturation, and the complement cascade [30].
The HLA recognizes foreign proteins (peptides) present in germs that enter the human body. If there is an interaction between HLA and peptide, the interaction formed will be brought to the cell surface and then recognized by T cells which will cause an immune reaction. HLA is highly selective and only binds to specific peptides, so it is important to predict the match between HLA protein and antigen peptide or T cell epitope so that their formation can trigger an immune response [31].
There are 6 T cell epitopes as a result of EIDB analysis of the L1HPV52 antigen. The T cell epitopes are YLQMASEPY, PYGDSLFFF, DSLFFFLRR, MFVRHFFNRA, IYYYAGSSR, and YYYAGSSSRL. These six epitopes were predicted can recognize several Indonesian HLAs class I (HLA-A*24:02, HLA-A*33:03, HLA-B*15:02). Observation on position of the T cell epitopes, especially on 3D structure of L1HPV52, some epitopes (number 1, 2, and 3) are located at/on the surface in both monomer and pentamer forms. Epitope number 4 is only on the surface when in monomer form. Meanwhile, epitope numbers 5 and 6 are located inside of the protein in form of monomers, pentamers, and VLPs. The epitope that can bind to Indonesian HLA class II (HLA-DRB1*12:02) is DSLFFFLRREQMFVRH.
The Indonesian HLA has similarities with HLA from South East Asia, especially for Java, Maluku, and Nusa Tenggara, namely HLA-B*15:02 and DRB1*12:02 (high frequency in Yogya). In addition, HLA class I related to Indonesians are A*24:07 (21.52%), A*33:03 (15.6%), A*24:02 (14.35%). While for Indonesian HLA class II were B*15:13 (11.18) and B*15:02 (11.6%) [32] [33].