Figure 3 : Amino acid sequence analyses, expression vector construction, and recombinant hTERT T-cell epitope expression.

(a) Published human MHC class I (HLA-A1, A2, A3, A24 and B7 allele) and class II (HLA-DR1, DR4, DR7, DR11 and DR15 allele and pan DR, DQ and DP) associated hTERT peptides are summarized. Sequences ofhTERT peptides were predicted using ANN [17] and SMM methods [18] and associations with mouse MHC class I and II of H-2d genotype with IC50 values of <150 nM are listed.
(b) The peptides hTERT602–641 and hTERT1088–1121 were selected and linked using an FFRK peptide to give hTERTepi. Subsequently, cDNA encoding the hTERTepi with codons optimized for E. coli was synthesized and inserted into the pET22b–PTD1J1(Tb) vector to express the recombinant PTD-J-hTERTepi protein.
(c) Recombinant PTD-J-hTERTepi was expressed in E.coli (T, total lysate) but was not found in the soluble fraction (S). The insoluble pellet was suspended in 6M guanidine–HCl (GT) and the insoluble fraction (GP) was removed by centrifugation. The supernatant fraction (GS) was applied to a Ni-NTA sepharose (fast flow, GE Healthcare) column and the recombinant protein was refolded and purified as described in the Materials and methods. Most recombinant protein was trapped in the column and little was observed in the flow-through fraction (F). No protein was found in detergent (D), cyclodextrin (C), orwash buffer (W) fractions. A few contaminating proteins were detected in the early elution fraction (E1) and pure recombinant PTD-J-hTERTepi protein was found in the peak elution fraction (E2). The gel was stained by Coomassie Blue R-250.

Chuang et al.Journal of Immunotherapy Applications  2015 2:1DOI : 10.7243/2055-2394-2-1