*2.2.1 DNA-interacting enzymes used and the mechanism of amplification reaction*

All the DNA-FACETM amplification-expression DNA vectors share variants of a universal DNA amplification module. The module may be custom modified and transferred into other DNA vectors, either prokaryotic or eukaryotic, containing alternative antibiotic resistance genes, origins of replication, transcriptional promoters, and translation initiation signals, among others. The amplification module contains two convergently oriented recognition sequences of the Type IIS REase - SapI, able to recognize asymmetric 7-bp 5′-GCTCTTC-3′ and cleave the upper DNA strand to the 3′ direction, at a distance of 1-nt and the bottom strand at a distance of 4-nt, thus leaving 3-nt 5′ cohesive ends. The SapI (and its isoschizomers) are unique among the discovered, atypical Type IIS REases. The SapI DNA recognition sequence is long and the protruding ends of the SapI cleaved substrate form a codon *DNA-FACE™ - An* Escherichia coli*-based DNA Amplification-Expression Technology… DOI: http://dx.doi.org/10.5772/intechopen.101640*

**Figure 2.** *Principles of the DNA-FACE™ technology.*

length upon ligation. The long DNA recognition sequence highly decreases the probability of its accidental and undesired appearance with both the amplificationexpression vector used and the DNA fragment to be amplified. Furthermore, the key feature of the SapI is a very rare occurrence of the 3-nt long protruding ends, that enable codon length formation in-between linked the amplified coding DNA segments, thus ensuring continuity of the ascending ORF. The SapI sites are separated by the orthodox Type II SmaI REase recognition sequence (5′-CCC|GGG-3′). SmaI cleaves its recognition sequence, leaving blunt ends. This is a convenient setup for cloning of any synthetic DNA fragments, as typically they are synthesized/delivered as double-stranded (ds) forms. The amplification-expression module provides three cloning options for a DNA fragment to be amplified (*i*) cohesive end cloning of the SapI generated 5′-CCC/5′-GGG sticky ends, (*ii*) blunt-end cloning of the SapI cohesive ends, previously filled in by T4 DNA polymerase/deoxyribonucleotides triphosphates; (*iii*) blunt-end cloning into SmaI site.

Whatever cloning option is used, the general protocol needs to be followed for all the amplification-expression vectors: (1) selection of bioactive peptides from a natural source or design of the monomeric DNA fragment to be amplified; (2) generation of the monomer by chemical synthesis of DNA, PCR amplification or REases excision from natural DNA; (3) cloning of the DNA fragment to the selected amplification-expression vector.

The cloning process is preferentially conducted using the cohesive end approach (mentioned above). The asymmetric 5′ -CCC/5′-GGG cohesive ends can be generated (a) through their addition at 5′- and 3′- termini of the dsDNA monomer during chemical synthesis or (b) *in vitro*, from SapI recognition sequences, added during chemical synthesis of the monomer and further clipped-off with SapI REase or (c) by PCR amplification with primers, containing SapI sites at their 5′- overhangs and clipping-off with SapI or (d) by excision of a SmaI-cloned monomer from an amplification-expression vector.

Subsequent stages of the DNA-FACE™ procedure include: (*i*) purification of the ds DNA fragment equipped with SapI-compatible 5′ -CCC/5′-GGG cohesive ends, ordered self-ligation of DNA monomers in directional, head-to-tail orientation, driven by asymmetric cohesive ends, (*ii*) ligation of generated concatemers mixture or of a selected gel-purified concatemer into the SapI-cleaved amplification vector, (*iii*) transformation into a suitable *E. coli* host strain, tolerant to atypical DNA sequences, such as DH5alfa, Top10, JM109, EnduraTM; (*iv*) selection of *E. coli* clones containing a concatemeric ORF segment with the desired number of monomers; (*v*) expression of the concatemeric ORF directly from an amplification-expression vector, containing strong transcription promoter, resulting in concatemeric protein biosynthesis or (*vi*) excision of the concatemer with SapI from the vector, which results in a DNA concatemeric segment equipped with SapI-cohesive ends and repeating steps (*i–iv*), until a desired number of monomeric DNA segments within a concatemer is obtained (**Figure 2**).

#### *2.2.2 Amplification-expression vectors*

Four categories of DNA amplification-expression vectors were designed for the purpose of the DNA-FACE™ technology: (I) pAMP series of six vectors for concatemeric protein biosynthesis in *E. coli* cytoplasm, (II) DNA amplification-expression pET21AMP-HisA vector for IPTG-controlled concatemeric protein biosynthesis in *E. coli* cytoplasm, (III) pET28AMP\_SapI-Ubq vector for cytoplasmic biosynthesis of concatemeric proteins fused with ubiquitin at N-terminus and (IV) pET28AMP\_ PhoA or pET28AMP\_MalE vectors for secretion of concatemeric proteins, produced under IPTG control, to the *E. coli* periplasm.

The pAMP series ((I); **Figure 3**) was constructed on the basis of the vector pACYC184 and its derivative pRZ4737 (W. S. Reznikoff) [5, 6, 30]. For the pAMP DNA vectors, six versions of the amplification modules were used, which differed by the presence/absence of His6\_tag for metal affinity chromatography in three different reading frames (**Figure 3**). The pAMP DNA vectors are compatible with the *colE1* origin vectors, such as pET-series, and can be maintained in the same *E. coli* cell, if needed [5–14].

The pET21AMP-HisA vector ((II); **Figure 4**) is based on the pET-21d(+) expression vector (Novagen, EMD Millipore Corporation).

The pET28AMP\_SapI-Ubq vector ((III); **Figure 5**) was designed as a modification of the pET-28d(+) expression vector (Novagen, EMD Millipore Corporation) and enables the fusion of a concatemeric protein with ubiquitin [5–7]. As concatemeric proteins contain repeated peptide modules, depending on their sequence, they may not form typical, natural protein structures, with hydrophobic amino acids residues forming a "core" surrounded by exposed polar and charged amino acids residues. This may affect their solubility, thus for some applications a fusion

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

#### **Figure 3.**

*The first set of amplification-expression modules designed for the DNA-FACE™ technology, used for the construction of the series of six pAMP DNA vectors (GenBank: MK606505, MK606506, MK606507, MK606519, MK606520, MK651654). All pAMP vectors are composed of: (a) p15A origin, (b) strong, temperature-regulated bacteriophage lambda pR transcription promoter, (c) bacteriophage lambda cI857ts repressor gene for control of pR promoter and host-independence, (d) an amplification module, containing two convergent SapI sites and a SmaI site and (e) chloramphenicol resistance gene.*

with ubiquitin may be beneficial. The ubiquitin domain can be removed from a fusion protein by deubiquitinating proteases [7, 31].

The pET28AMP\_PhoA or pET28AMP\_MalE vectors ((IV); **Figure 6**) contain two alternative DNA segments, coding for *E. coli* secretion leaders MalE or PhoA. The MalE/PhoA encoding DNA segments are located at the 5′ end of the fused ORFs.

Detailed protocols, maps, sequences of all the amplification-expression vectors series have been published elsewhere [5–7].
