*2.3.1 Testing the DNA-FACE™ with model HBV antigen S epitope and construction of functional concatemeric proteins*

To evaluate the theoretical assumptions made during DNA-FACE™ biotechnology design, a 7-aa HBV epitope derived from S protein was selected [5]. The *E. coli*

#### **Figure 4.**

*The pET-derived, amplification-expression DNA vector, designed and constructed for the DNA-FACE™ technology. The pET21AMP-HisA DNA vector (GenBank MK606521) contains (a) colE1 origin, (b) f1/M13 origin, (c) very strong T7-lac transcription promoter, inducible by lactose or IPTG, (d) amplification module HisA and (e) ampicillin resistance gene.*

expression optimized synthetic 21-bp DNA fragment, encoding the epitope TKPTDGN was cloned into the amplification-expression pAMP1-HisA vector. The detailed procedure of cloning of the synthetic 21-bp DNA fragment and its further amplification (based on the DNA-FACE™ technology) was described by Skowron et al. [5]. The results are shown in **Figure 7** (see the first round of amplification).

Further, the selected 5-mer was subjected to the second round of amplification (**Figure 7**; see the second round of amplification). The amplification-expression vectors were designed in such a way, that no SapI recognition sites were left within the amplified DNA segment. Such a vector design makes it possible to use a multimeric DNA fragment, obtained in the first round, as a "monomer".

Subsequently, an alternative or hybrid route of a DNA fragment amplification was tested. A possibility of a combination of chemical synthesis of the pre-formed HBV epitope-coding DNA, pushed to its technical limits with DNA-FACE™ method, was investigated. The limits of such chemical synthesis strongly depend on the DNA sequence and the size of the DNA fragment to be concatemerized. The model HBV epitope coding DNA turned out to be a rather "friendly" case, as compared to the other designed DNA sequences. Testing several commercial services, chemically synthesizing the designed DNA molecules, a maximum of 25-mers within a single synthetic gene was obtained. The 25-mer was then used as a "monomer" in DNA-FACE™ biotechnology amplification. As a result, bacterial clones

*DNA-FACE™ - An* Escherichia coli*-based DNA Amplification-Expression Technology… DOI: http://dx.doi.org/10.5772/intechopen.101640*

#### **Figure 5.**

*The pET-derived pET28AMP\_SapI-Ubq DNA vector, designed and constructed for the DNA-FACE™ technology. The pET28AMP\_SapI-Ubq DNA vector (GenBank MK606527) is composed of: (a) colE1 origin, (b) f1/M13 origin, (c) T7-lac transcription promoter, inducible by lactose or IPTG, (d)) the DNA fragment amplification module His6\_c-Myc\_WYY\_ubiquitin\_SapI-Sma-SapI, enabling ubiquitin gene fusion and (e) kanamycin resistance gene.*

containing up to 500-copies of the 21-bp HBV epitope were obtained (**Figure 7**; see the alternative round of amplification [5, 7–14].

During the next stage of the technology testing, the ability of the amplificationexpression vector to yield an efficient translation of a highly atypical concatemeric gene, was investigated. The selected pAMP-HisA constructs, exemplified by 10-mer, 13-mer, 15-mer, 20-mer and 30-mer, were expressed (**Figure 8**). The expression of the recombinant constructs with up to 450 repeats, (composed of the 7-aa monomers), was tested.

It is known that the upper limit of molecular weight of a single polypeptide, biosynthesized by *E. coli,* is app. 150–200 kDa. However, due to a potential "slippage" of the translation machinery on mRNA repeat and a possible premature translation termination, a "smear" on SDS-PAGE gels was typically observed. The "smear" was located near the expected position of the recombinant protein, with the size corresponding to its molecular weight (**Figure 8**) [5]. Nevertheless, even a mixture of translation products is expected to be fully functional in planned applications, as each monomeric unit is semiindependent in genetically programmed functions, such as comprising an immunologically condensed "artificial" antigen, built from immunoactive epitopes only.

Afterwards, the DNA-FACE™ biotechnology was validated in the construction of prospective, pro-regenerative drugs and in the concatemeric proteins designed for remediation of the environment and new generation biosensors. Taken together, over 50 concatemeric ORFs and the resulting concatemeric proteins were constructed. Among those, a series of prospective pro-regenerative drugs was developed [5, 6]. For this purpose, the amplification (concatemerization) of four types of the designed DNA fragments was performed. The selected DNA fragments encoded either the laboratory-developed/predicted peptides or the peptides originally derived from wound healing stimulatory proteins.

The first selected peptide -TSRGDHELLGGGAAPVGG, which originated from the angiopoietin-related growth factor (AGF), was used for the construction of a poly-signal protein [5]. The peptide was linked to the elastase recognition sequence

#### **Figure 6.**

*The pET-derived, amplification-expression-secretion DNA vectors: pET28AMP\_PhoA and pET28AMP\_MalE, designed and constructed for the DNA-FACE™ technology. The pET28AMP\_PhoA DNA vector (GenBank MK606526) and the pET28AMP\_MalE vector (GenBank MK606522) contain: (a) colE1 origin, (b) f1/ M13 origin, (c) T7-lac transcription promoter, inducible by lactose or IPTG, (d) the amplification module His6\_PhoA\_SapI-Sma-SapI or His6\_MalE\_SapI-Sma-SapI, (e) kanamycin resistance gene.*

to facilitate a gradual enzymatic release of the monomers/oligomers, cleaved by the elastase present in human serum [5, 6]. The AGF is known to promote epidermal proliferation, new blood vessel formation, and wound healing in the skin.

The second type of the selected peptides – the RGD and RGDGG peptides – originated from fibronectin. These motifs function as crucial cell-binding factors. *DNA-FACE™ - An* Escherichia coli*-based DNA Amplification-Expression Technology… DOI: http://dx.doi.org/10.5772/intechopen.101640*


#### **Figure 7.**

*The DNA-FACE™ technology proof of concept – amplification of the model epitope encoding DNA fragment. The first round of amplification: the synthetic DNA fragment, encoding the model epitope, was cloned into the amplification-expression pAMP1-HisA vector as described by Skowron et al. [5]. Then, the PCR amplification of the appropriate DNA segment was performed. The PCR product was cleaved with SapI and subjected to autoligation at 16°C using T4 DNA ligase and aliquots were taken at intervals of 5, 10, 20, 40, 80, and 160 min. A series of DNA segments of increasing length was obtained. The resulting concatemers were pooled and cloned in pAMP1-HisA, cleaved with SapI. The obtained bacterial clones were analyzed by colony PCR [5]. The second round of amplification: the selected 5-mer was subjected to the second round of amplification [5]. The appropriate DNA fragment was excised from the E. coli clone plasmid and subjected to autoligation. The reaction products were pooled, ligated back to the pAMP1-HisA. The plasmid DNAs from the positive bacterial clones were cleaved by SapI and the obtained restriction fragments were analyzed electrophoretically. Alternative round of amplification: the synthetic 25-mer was subjected to the amplification as described by Skowron et al. [5]. The autoligation products were ligated to the pAMP1-HisA. The plasmid DNAs from the positive bacterial clones were cleaved by SapI and the obtained restriction fragments were analyzed electrophoretically.*

#### **Figure 8.**

*Concatemeric, recombinant proteins consisting of multiple repeats of a model HBV epitope, obtained with the DNA-FACE™ technology.*

The RGD sequence is present in several extracellular matrix (ECM) molecules and is responsible for the mediation of cell attachment. It is known to promote cell/tissue interaction with artificial biomaterials and shows a pro-regenerative effect [32, 33].

The third designed peptide - RLIDRTNANFLGGGAAPVGGG originated from the platelet-derived growth factor (PDGF B). PDGF B functions as a mitogen for fibroblasts and smooth muscles cells and regulates embryonic development. The peptide was extended by GG helical breakers and an AAPV peptide, known to be effectively cleaved by human elastase [34–36].

The fourth peptide series: GHK, GHKGG, GHKGGGAAPVGG, KGHKGGGAAPVGG was designed on the basis of the GHK peptide, which naturally occurs in human plasma and can be released by the injured tissues. The peptide is responsible for diverse protective and healing actions. For example, it is known to improve tissue repair, stimulate blood vessel and nerve outgrowth, boost collagen, elastin, and glycosaminoglycan synthesis [37, 38].

Another explored application of the DNA-FACE™ biotechnology includes construction and evaluation of concatemeric proteins for the purpose of environment remediation and biosensors development. These proteins target toxic heavy metal ions: Pb2+, Hg2+ Ag+ , As3+, Ni2+, Cd2+, and the uranyl ion (UO2 2+). The details will be released to public domain following submission of the patent application.

#### *2.3.2 Final note concerning concatemeric proteins constructions*

It should be noted that the maximum possible monomer copy number within a constructed DNA concatemer could be lower or higher than in the case of the model *DNA-FACE™ - An* Escherichia coli*-based DNA Amplification-Expression Technology… DOI: http://dx.doi.org/10.5772/intechopen.101640*

HBV epitope [5], as it strongly depends on the DNA sequence and the length of the DNA fragment to be concatemerized. Further precautions concern the downstream applications of the DNA concatemers. Namely, some of the constructed concatemeric genes/ORFs may not be efficiently or error-free transcribed or expressed in *E. coli*. This is again highly dependent on the nt sequence of the resulting mRNA as well as on its resistance to form stable secondary and tertiary structures, which may hide the translation initiation/termination signals or stall the translating ribosomes. Moreover, certain translated amino acids sequences, especially these appearing in ascending concatemeric proteins as repeated segments, may yield low expression levels due to the depletion of highly used aminoacyl-tRNAs, as well as cause the insolubility or toxicity to the recombinant host. Although these potential problems generally concern recombinant genes expression, they may be more pronounced due to the "artificial" nature of the concatemeric proteins. If needed, an implementation of additional strategies can be helpful, such as testing various cultivation/ expression conditions, fusions with non-concatemeric proteins, *E. coli* strains, alternative prokaryotic or eukaryotic expression systems, among others. It is worth noting that the DNA amplification-expression modules can be easily transferred to other DNA vectors, including eucaryotic, if necessary.
