Construct design - 'Think before you ink'

While think before you ink is a popular slogan by tattoo adversaries, it has also been 100% applicable to creating our approach of bioprinting. Careful planning is, of course, necessary to create a construct that fulfills its role in the most functional way possible. In the following, we are going to explore the thought processes that went into the design of the receptors we built to mediate cross-linking of cells for bioprinting, as well as the design of other functional parts involved in our project.

The eukaryotic receptor

Schematic depiction of the expression vector for the BAP-based receptor as well as the corresponding biotin ligase. Shown are the CMV promoter element, the signal peptide (depending on the construct being the hEGFR signal peptide, the IgKappa signal peptide or the BM40 signal peptide, the latter two further- more possessing an elongated 5’ UTR), the biotin acceptor peptide, a nanoluciferase, an epitope tag for A3C5 antibodies, the hEGFR N-terminal transmembrane alpha-helix, mRuby, a Strep-tag II, an IRES from eIF4G1 for polycistronic transcription, BirA including a IgKappa signal peptide as well as an ER retention signal sequence, and the hGH polyadenylation signal sequence.Terminator elements are not shown. Please note that proportions used in the illustration do not resemble actual sequence lengths.

The receptor we created is able to cross-link cells after injection into streptavidin solution by allowing the connection of several biotinylated cell surfaces through the tetravalent binder streptavidin. This reaction being very fast and highly affine - with a K_D of 10^-15 M^-1 - allows precise positioning and cross-linking of cells for the creation of cellular matrices by bioprinting. For this project, we have created receptors for cross-linking of both human as well as bacterial cells.

The functionality of the receptor is mediated by an extracellular biotin acceptor peptide (BAP) that is endogenously biotinylated by a coexpressed biotin ligase (BirA). The biotin ligase is therefore encoded by the same plasmid as the receptor, with an internal ribosome entry site (IRES) taken from the human eukaryotic translation initiation factor 4G1 gene (eIF4G1) allowing translation of two open reading frames (ORFs) from a single polycistronic plasmid. BirA is targeted to the ER via an Igkappa signal peptide as well as an ER retention signal (a so-called KDEL sequence). Thus being permanently located in the ER, BirA is able to biotinylate the BAP of translocating proteins upon their translocation, i.e. to the cell surface.

For the receptor itself, it is of course of utmost importance that it is correctly anchored in the membrane, resulting in the correct localization of domains in either intercellular or extracellular space. For this purpose, a transmembrane domain of the human epidermal growth factor receptor (EGFR) was chosen. For correct trafficking of the receptor into the ER and, subsequently, its relocation on the cell surface, a properly functioning signal peptide had to be determined. More details on the decisions involved in protein targeting can be found in the corresponding section ('Targeting').

Moreover, the receptor contains three functional elements for its detection: An intracellular red fluorescent protein (mRuby) for detection of the receptor via fluorescence microscopy, an extracellular nanoluciferase for detection via luciferase assays and an extracellular epitope domain for detection via A3C5-monoclonal antibodies (which may be coupled to secondary conjugates for detection via immunofluorescence or which may be used for immunoprecipitation). For purification of the receptor, it furthermore contains a C-terminal Strep-tag II for purification via streptactin affinity chromatography.

The vector furthermore contains the poly-adenylation signal of human growth hormone (hGH) for functional polyadenylation of the transcribed mRNA. A schematic depiction of the vector is shown below. A description for the respective functional elements is given in the following sections.

The biotin acceptor peptide (BAP)

The biotin acceptor peptide is a short peptide sequence originating from E. coli. The sequence, being composed of 15 amino acid residues, is biotinylated specifi- cally at a lysine residue within the recognition sequence by a coexpressed biotin ligase (BirA) and thus mediates the functionality of the receptor.

EGFR transmembrane domain

The N-terminal transmembrane alpha-helix of human EGFR (UniProt P00533, amino acids 622-653) was chosen as the transmembrane domain of the receptor. A stop-transfer sequence consisting of charged amino acids was added at the C-terminus, and the sequence was furthermore flanked by a (GGGGC)2-linker at the N- and C-terminus, respectively. As predicted by the TMHMM 2.0 server for the prediction of transmembrane helices, amino acid residues N-terminal of the transmembrane domain are positioned outside of the cell, while residues C-terminal of the domain are positioned on the inside of the cell.</p>

Schematic depiction of the vector encoding the cell surface receptor for cross-linking of human cells.

A3C5 epitope tag

Antibodies have various areas of application in life science, including protein detection, pulldown experiments and even immunotherapy. Having been discovered in 1995 during an in vitro screening for antibodies against cytomegaloviral proteins,14 the A3C5 antibody is an established molecular tool for specific recog- nition of proteins. By tagging cell surface proteins with an epitope specifically recognized by A3C5 (being a minimal peptide sequence of 11 amino acids), one is easily able to detect tagged proteins via immunofluorescence microscopy, FACS or one may purify them via pulldown experiments. Through the latter, one may also screen for in vivo interaction partners of the tagged protein.

Nanoluciferase

Fireflies have fascinated mankind for millenia. The concept of bioluminescence indeed has a great meaning for several invertebrate species, allowing communication with other individuals, signaling receptiveness or deterring predators. The most common enzyme responsible for the creation of bioluminescence are the luciferases (from the latin words ’lux’ and ’ferre’, meaning ’light-carrier’). These enzymes, among others found in fireflies and deep-sea shrimp, commonly consume a substrate called luciferin as well as energy in the form of ATP or reduction equivalents in order to create light-emission through oxidation. The underlying mechanism hereby relies on the creation of an instable, highly excited intermediate under the consumption of energy. This high-energy intermediate then spontaneously falls back into a state of lower energy, emitting the energetical difference as visible light.

Schematic depiction of the vector encoding the cell surface receptor for cross-linking of human cells.

Luciferases have also found their way into biotechnological applications. The simplistic concept of creating visible (and thus easily measurable) light makes luciferases ideal reporters for the expression of proteins via a corresponding assay. This project also makes use of a luciferase for the determination of expression levels of the surface receptor. The monomeric luciferase used in this project, the so-called ’NanoLuc-RTM-' (due to terms of simplicity referred to as ’nanoluciferase’ in the following), can be fused to other proteins in order to make their expression visible and quantifiable. This engineered luciferase emits a steady, easily detectable glow when exposed to its substrate, while being only 19 kDa large and being brighter, more specific and steadier than other luciferases commonly found in nature. Using luciferases offers several advantages over fluorescent proteins, including smaller size and decreased damage to cells during measurements, as no excitation is necessary for light emission.

mRuby

Having been engineered as a monomeric form of the red fluorescent protein eqFP611, mRuby is one of the brightest red fluorescent proteins available. With an excitation maximum at a wavelength of 558 nm and an emission wavelength maximum of 605 nm, the resulting stokes shift of 57 nm makes mRuby a good choice for fluorescent microscopy imaging and, thus, allows the visualization of the receptor in its cellular environment by being fused to the C-terminus of the transmembrane domain. mRuby is especially powerful for the fusion with receptors - not only due to its small size and high stability, but also due to the fact that, in comparison with eGFP, it appears up to 10 times brighter in membrane enviroments.

Strep-tag II

The Strep-tag II is an eight amino acid peptide sequence that specifically interacts with Streptavidin and can thus be used for easy one-step purification of the receptor via affinity chromatography.

CMV-promoter

If much is good, more must be better. Last but not least, we of course need our receptor to be expressed to a bigger extent for efficient cross-linking, so that a high avidity for the interaction may be achieved. The CMV promoter (taken from the parts registry, BBa_K74709) enables constitutively high expression and thus suits our needs perfectly.