DNA-FACE™ (DNA Fragment Amplification & Concatemeric Expressed Nucleic Acids and Proteins) is a universal technological platform, created with the use of molecular biology methods, based on the amplification of specific DNA fragments for the construction of targeted biomolecules. DNA-FACE™ is applicable to the development of vaccines, programmed proteins, drugs and industrial and environmental processes.
DNA-FACE™ technology is protected worldwide by 5 granted patents and 6 patent applications. This break-through biotechnology of ordered DNA fragment amplification-expression and construction of genetically programmable arficial proteins provides scientfic and economical solutions, not possible with other known technologies.
Figure 1. Diagram showing the principle of the developed DNA fragment amplification-expression method.
Figure 2. Results of the first round of amplification of the model 7-aa HBV epitope. Panel A. Electrophoretic analysis of the DNA autoligation products. Lane K, non-ligated DNA insert encoding one HBV epitope; lanes 1-6, DNA autoligation products after incubation with T4 DNA Ligase for 5, 10, 20, 40, 80 and 160 min, respectively; lane 7, a mixture of all the DNA autoligation products from lanes 1-6; lane M, Sigma PCR 20-bp Low Ladder (selected bands marked). Panel B. PCR amplification of the obtained artificial genes encoding repeats of the HBV epitope. Lane M1, GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher Scientific); lane M2, GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific); lane K1, control PCR fragment 230 pb; K2, control PCR fragment (template: pAMP1-HisA); lanes 1-10, colony PCR fragments (templates: pAMP1-HisA recombinant constructs, encoding 1, 2, 3, 4, 5, 6, 7, 8, 10 and 13 repeats of the HBV epitope, respectively. Panel C. Biosynthesis of the recombinant 13-mer in E. coli BL21(DE3). The proteins were separated by SDS-PAGE and stained with Coomassie Blue R250. Lane M, protein ladder (LMW Calibration Kit, GE Healthcare); lanes 1-4: recombinant E. coli culture, containing a 13-mer HBV construct prior to induction, and sampled at 0, 1, 2 and 3 h. lanes 5-9: recombinant E. coli cultures, containing the 13-mer HBV construct after thermal induction, sampled at 1, 2, 3, 4 and 16 h. The characteristic pink-violet protein band representing the poly-HBV epitope protein (13-mer) is marked with an arrow. Panel D. Purification of the recombinant poly-HBV epitope protein (13-mer; 13.1 kDa). Lane M, Protein Ladder (GE Healthcare); lane 1, after CM Sepharose chromatography; lane 2, after Immobilized Metal Affinity Chromatography (IMAC); lane 3, after Resource S cation exchange chromatography; lane 4, after size exclusion chromatography (SEC) - fraction of the protein with lower molecular weight; lane 5, after SEC – fraction of the protein with higher molecular weight.
Figure 3. Results of the second round of amplification of the 5-mer of the 7-aa HBV epitope. Panel A. Electrophoretic analysis of the DNA autoligation products. Lane M, GeneRuler 100 bp Plus DNA Ladder (Thermo Fisher Scientific); K, non-ligated DNA insert encoding concatemeric protein containing five repeats of the HBV epitope; lanes 1-6, DNA autoligation products after incubation with T4 DNA Ligase for 5, 10, 20, 40, 80 and 160 min, respectively; lane 7, a mixture of all the DNA autoligation products from lanes 1-6. Panel B. The predicted restriction fragment lengths following excision of the inserts with SapI. Panel C. SapI restriction analysis of the obtained recombinant pAMP1-HisA constructs encoding polyepitopic proteins of different length. Lane 1, pAMP1-HisA_HBVep_10; lane 2, pAMP1-HisA_HBVep_15; lane 3, pAMP1-HisA_HBVep_20; lane 4, pAMP1-HisA_HBVep_30; lane M, GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific). Panel D. Purified recombinant polyepitopic protein variants after HiTrapTM IMAC FF chromatography. The proteins were separated by SDS-PAGE and stained with Coomassie Blue R250. Lane M, protein ladder (LMW Calibration Kit, GE Healthcare); lane 1, the 10-mer of the HBV epitope (10.5 kDa); lane 2, the 15-mer (14.9 kDa); lane 3, the 20-mer (19.2 kDa); lane 4, the 30-mer (28.7 kDa).