A simpler and more cost-effective peptide biosynthetic method using the truncated GST as carrier for epitope mapping

Abstract

There is a need to develop better methods for epitope mapping and/or identification of antibody-recognizing motifs. Here, we describe improved biosynthetic peptide (BSP) method using a newly developed plasmid pXXGST-3 as vector, which has a viral E7 gene in the cloning sites of pXXGST-1. It is crucial to employ pXXGST-3 instead of pXXGST-1, since it makes use of the BSP method simpler and easier to perform, and more cost-effective for epitope mapping. These merits are embodied in two aspects: i) convenient recovery of double enzyme-digested product due to the existence of 315 bp inserted between BamH I and Sal I sites, and thus greatly reducing the production of self-ligation clones, and ii) no longer requiring control protein when screening recombinant (r-) clones expressing 8/18mer peptides by running polyacrylamide gel electrophoresis. The protocol involves the following core steps: (i) design of plus and minus strands of DNA fragments encoding overlapping 8/18mer peptides; (ii) chemical synthesis of the designed DNA fragments; (iii) development of r-clones using pXXGST-3 vector expressing each 8/18mer peptide fused with truncated GST188 protein; (iv) screening r-clones by running the cell pellets from each induced clone on SDS-PAGE gel followed by sequencing of inserted DNA fragments for each verified r-clone; and (v) Western blotting with either monoclonal antibodies or polyclonal antibodies. This improved GST188-BSP method provides a powerful alternative tool for epitope mapping.

Epitome mapping of E6 and E7 proteins from high-risk oncogenic HPV types 16, 18 and 45

“…Clearly, the key to achieve this goal is that entire IgG-epitomes of several target viral proteins and all type-specific BCEs in them can be revealed. Now, it is achievable since all linear BCEs present in a protein and type-specific BCEs in them has been able to be delineated using rabbit/murine pAbs using the BSP method specific for BCE mapping [35,36], which has been used in many studies of mapping BCEs on different viral protein using rabbit pAbs and BSPs [30,[48][49][50][51][52]. Our previous study has revealed three IgG-epitomes of E6, E7…”

“…To our knowledge, the BCE minimal motifs mapped using mAbs and chemically synthesized peptides (CSPs) together with ELISA were 4mer-6mer peptides [24,28,29,54], whereas the BCE motifs mapped using pAbs and BSPs together with Western blotting can be the shortest 3mer-peptides [30,55] and the longest 9mer-peptide (18/E6-4) mapped in this study, even longer 10mer-peptide [52]. In addition to contributing to reveal complete BCEs on a protein using animal sera and their specificity among homologous proteins, the BCE motif identification has two other implications: i) helping to the design of multi-BCE peptide diagnostic reagents and/or vaccines, which would improve the accuracy and sensitivity of serological diagnosis by combining many specific BCEs as possible, and prevent potential harmful antibody cross-reactivity to other key proteins in human body, that is, it should avoid using the mapped 3mer and 4mer-peptide BCEs if they are used as candidates of preventive vaccine; and ii) conducing to understand the possible regular differences of BCE motifs located the same site that are mapped by various species pAbs and/or mAbs, for instance, the PTRR and ELRHY motifs [29,36] mapped by mAbs E6-18-1 and C1P5 are less one residue at the N-terminal of them than the DPTRR and RELRHY motifs of 18/E6-1 and 18/E6-2 mapped by rabbit pAbs. Similarly, the delimitated HR-BCE h-16E6-1 motif is less one residue at its C-terminal compared with the RR-BCE 18/E6-5.…”

“…The molecular cloning of synthesized DNA fragments was done as described previously [36], which is briefly described as follows: i) designing all plus and minus strands of DNA fragments encoding each designed 8/16mer-peptides of the target proteins, having cohesive ends for BamH I and Sal I restriction enzymes at their 5' and 3' ends; ii) making 100 μM stock solution of each synthesized DNA fragment by adding appropriate volume of double distilled H 2 O (ddH 2 O) into tubes, respectively; iii) taking respectively 5 µl of the stock solution from two tubes in pairs into a 1.5 ml of new tube, and add 90 µl of ddH 2 O to make 5 μM final concentration of each DNA fragment; iv) heating tubes up to 94ºC in an electric heating block, anneal for 5 min, and then allowed them to cool gradually to room temperature (RT); v) performing the ligation reactions using above each annealed DNA fragment and vector of pXXGST-3 digested with BamH I and Sal I enzymes at 16ºC overnight; vi) using the above ligation mixture in each tube to transform the E. coli BL21 (DE3) competent cells, and growing overnight each clone on solid Luria Broth (LB)-ampicillin (amp) plates at 37ºC; vii) growing overnight each clone from LB-amp plates in 3 ml of liquid LB-amp medium at 30ºC, and repeating once after dilution 1:50 in fresh LB-amp medium to an OD 600 of 0.6-0.7. The clones were induced at 42ºC for 4-5 h; viii) harvesting each cell pellets by centrifugation, and then running sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) to screen r-clones according to the specific band of 8/16mer-peptide fusion protein shown on the gel; ix) conducting DNA sequencing of all synthesized DNA fragments inserted into plasmid pXXGST-3 from r-clones verified by SDS-PAGE analysis.…”

“…The designed overlapping 16mer/8mer-peptides for two rounds of antigenic peptide and fine BCE motif mapping of E6 and E7, which all had an overlap of 8 and 7 aa residues respectively, were expressed as GST188 fusion proteins as described earlier [36]. Briefly, the synthesized annealed DNA fragments encoding each 8/16mer-peptide, incorporating BamH I and TAA-Sal I cohesive end on their 5′and 3′ends, were inserted into the polyclonal region downstream of GST188 gene in pXXGST-3 plasmid; BL21 (DE3) pLysS E. coli cells were transformed with r-plasmids; respective two or three clones grown on the LB-amp plates were picked-up to conduct thermo-inducible expression and the harvested cell pellet was first used to run 15% SDS-PAGE to determine respective r-clones according to the specifically expressed short peptide fusion proteins with about 23 and 24 kDa, since there is no any visible specific band in the weak antigenic area of proteins from non-recombinant clone.…”

“…It had been a challenge to delineate all fine BCEs on target viral proteins by rabbit and murine pAbs, including followed by determining HPV type-specific and conservative BCEs of them. In previous studies, using the biosynthetic peptide (BSP) method with simple, economic and reliable merits [35,36], we have decoded three complete IgG-epitomes of oncogenic E6, E7 and major capsid L1 proteins from hr-HPV58 with rabbit pAbs raised against respective r-proteins. The specificity and conservativeness of each mapped BCE among defined and possible hr-HPVs were determined via homologous sequence alignments based on BCE minimal motif.…”