Construction of the plasmid pABA
The sequence of the gene expressing the E7 mutein assembled by polymerase chain reaction (PCR) is shown in the Figure 1A. The recognition sites for HindIII and BamHI restriction enzymes are included at the beginning and at the end of this sequence, respectively. In addition, two replacements by glycine were done, both contained in the L25X26C27X28E29 domain. Mutations in this domain avoid E7 protein interaction with the retinoblastoma protein (pRb), being a regulatory advantage for the future development of a therapeutic vaccine based on the E7 oncoprotein (10, 11). The band corresponding to the designed E7-18m gene had the expected size of 327 bp, as shown in Figure 1B.
After enzymatic digestion of the vector pPEPE7M-7 and following insertion of the E7-18m gene, the E7 mutein was fused to the CPP CIGB550 obtaining the CIGB550-E718 gene into the new vector denominated pABA (Figure 2). Thirteen clones were selected, with twelve clones being positive to restriction analysis. These clones were sequenced and three out of 12 clones had the sequence corresponding to the recombinant protein CIGB550-E718 (Figure 2). The clone number two was selected for following assays.
Expression of the fusion protein CIGB550-E718
E. coli BL21(DE3) and BL21-RP cells were transformed with the pABA vector and the expression of the recombinant protein was induced. The protein expression analysis by 15% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) was made for both strains revealing a band at the expected molecular weight (MW) of approximately 20 kDa, which corresponds with the recombinant protein CIGB550-E718 (Figure 3A).
The expression of the CIGB550-E718 accounted for approximately 14.7% of the total cellular protein in BL21(DE3) strain compared to only 4% in BL21-RP cells. Higher MW aggregates and a degradation of this protein were visualized by the Western Blot analysis (Figure 3B).
Bl21(DE3) strain transformed with pABA plasmid was selected to evaluate the expression of the CIGB550-E718 in different culture media using two carbon sources: glucose and glycerol. As shown in Figure 4 panels A and B, the major band of 20 kDa was observed for all culture media evaluated using any of the carbon sources. In comparative terms, the best protein expression was achieved when glycerol was added to the culture media.
The protein expression determined by densitometry was approximately 16% of total cellular protein in the M9 salt medium enriched with casein hydrolysate and glycerol (medium identified with the letter D). Statistical analysis (Figure 5) for each condition suggests that there are no significant differences among culture media containing glycerol (p>0.05). However, for culture media containing glucose, the expression of the recombinant protein was higher for the culture medium containing only M9 salt (identified with letter A) respect to the same medium enriched with tryptone, yeast extract and casein hydrolysate (p<0.05). Thus, taking in consideration these analyses, M9 salt medium enriched with casein hydrolysate and glycerol as a carbon source (medium identified with the letter D) was selected for protein purification process.
Purification of the recombinant protein CIGB550-E718
The non-optimized purification process of the CIGB550-E718 protein was carried out from 2.5 L of culture medium containing M9 salt, casein hydrolysate and glycerol (medium identified with letter D). About twelve grams of biomass per liter of induced culture were obtained at the end of the fermentation process.
As shown in Figure 6A lane 2, the CIGB550-E718 accounted for about 16% of the total cellular protein, and migrates as an approximately 20 kDa protein in 15% SDS-PAGE. This molecule was found in the insoluble fraction after cell disruption (Figure 6A, lane 3). Therefore, a protein extraction step using 6 M urea was done in order to obtain the protein in the soluble fraction (Figure 6A, lane 6). The following phase consisted in a single chromatographic step of IMAC which allowed us to obtain the recombinant protein CIGB550-E718 with 90% purity (Figure 6A, lane 10).
The immunoidentification analysis using an anti-HPV-18 E7 polyclonal antibody is shown in Figure 6B. A major band of approximately 20 kDa and aggregates of the CIGB550-E718 are revealed from eluates of IMAC and molecular exclusion (Figure 6B, lanes 9 and 10).
Yields of the purification process were around 32 mg of the recombinant protein CIGB550-E718 per liter of induced culture.