E.Coli derived camelid antibodies as a sensor for p53 in saliva

To date, around 35 mAb-s are approved for therapeutic applicationsby European Medicines Agency and US FDA. Most of the Ab-s are chimeric or humanized, and only a fraction are next-generation Ab fragments. Growing interest is received by next-generation Ab fragments such as Ab-s containing only antigen-binding fragments (Fab-s), single chain variable fragments (scFv-s), and heavy-chain Ab-s (HcAb-s). There are many advantages of using smaller fragments for therapeutic or diagnostic purposes: faster and cheaper engineering, improved tissue penetration and decreased immunogenicity.

Our team investigatedabout heavy-chain only Ab-s. HcAb-s are found in sera of camelids such as llamas or camels. They do not possess the first constant domain CH1, and have variable domains referred to as VHH-s or nanobodies. Nanobodies are the smallest naturally occurring fragments (around 120 aa) with the capability of binding antigens. The structure of a nanobody is highly advantageous: it is small and soluble, it is highly stable, and binds to a target with high specificity and affinity. The main structural difference of nanobodies is the presence of several amino acid signatures in the solvent-exposed surfaces, which are typically covered by VL-s of conventional Ab-s (Figure 1). Also, nanobody domains have a markedly longer CDR3 loop; that should compensate for the absence of a light chain (specifically VL), and it is highly possible to increase nanobody solubility by shielding hydrophobic regions in FR2. Expression of a VH domain of a conventional Ab with no light chain was found to be inefficient due to aggregation, low solubility and stickiness.

Figure 1.Schematic representation of differences in amino acid sequence between cDNA encoding variable regions of regular and heavy chain antibodies. The critical amino acid substitutions in VHH are marked by lightning symbols. Thick black lines connecting FR1-FR3 and CDR1-CDR3 represent disulfide bridges that stabilize the structure of the heavy chain

Nanobodies can be conjugated to other proteins without loss of function. They can be expressed and secreted in many organisms including E.coli in high amounts, which reduces the cost of production. Based on available sequences of nanobodies, our team designed a biosensor to test saliva samples for p53 protein, which is a biomarker for oral squamous cell carcinoma (OSCC).Elevated levels of p53 protein can beobserved in OSCC patients at different stages of the disease.

The test is similar to ELISA. The biosensor is a homodimer that constitutes VHp53, zipper-containing polypeptide, His-tag, red fluorescent protein (RFP) and HlyA that is a C-terminal signal sequence of alpha-hemolysin type I transport system. The construct along with HlyB and HlyD of alpha-hemolysin is expressed in E. coli. Zipperprovides dimerization capacity of separate chains, and hemolysin secretion system ensures the secretion of nanobodies from E. coli. Purified nanobodies are used for detection of p53 protein is saliva samples.

Introducing competence into E. coli using phi 29 DNA packaging motor

We became interested in this topic because E. coli is one of the most popular model organisms used for laboratory work. However, E. coli cells possess a weak tendency to uptake DNA from the surroundings, i.e. it shows a weak competence. As you know, competence can be induced by two common methods: electroporation and chemical transformation using divalent cations. Electroporation requires extra equipment as electroporator, while chemical transformation takes at least one day preparation. Therefore, we suggest to incorporate hexameric ATPasegp 16 from DNA packaging motor of phi29 virus into inner and outer membrane of E. coli using signal leading peptides from the iGEM distribution kit.

DNA packaging motor. This protein can be found in virus phi29 from B. subtilus. The protein belongs to superfamily of AAA+ motors (ATP-ases associated with diverse cellular activities). Gp16 functions as ATPase during DNA packaging. It has been observed that gp16 binds DNA without sequence specificity, but specifically to pRNA,which implies that the binding properties of gp16 are important determining factors for the biological activity of gp16.

The advantage of phi29 DNA packaging motor is that has a larger channel diameter, and hence it can easily translocate both dsDNA and ssDNA and large cavity size of this channel enables relatively easy insertion of functional moieties with desirable characteristics via conjugation to amino acid residues lining channel cavity or at terminal ends for diverse applications. However, the disadvantage of this protein for our idea is that it can transport only linear DNA.

Our aim:To express gp16 of DNA packaging motor from phi29 on the surface of E. coli and to determine the uptake of DNA plasmids by the modified bacterium.

References

Smolarek, D., Bertrand, O., & Czerwinski, M. (2012). Variable fragments of heavy chain antibodies (VHHs): a new magic bullet molecule of medicine?. Advances in Hygiene & Experimental Medicine/Postepy Higieny i Medycyny Doswiadczalnej, 66.

Schwartz C, De Donatis GM, Fang H, Guo P. (2013). The ATPase of the phi29 DNA packaging motor is a member of the hexameric AAA+ superfamily. Virology. 443: 20–27.