Human collagen is a complex protein to produce recombinantly. The tight left-handed helix structure is facilitated by numerous hydroxyproline residues¹ which must be produced by post-translational modification by an enzyme with complex quaternary structure² with the function only demonstrated natively in a single relatively common yeast strain, Ogataea polymorpha.³ The traditional collagen motif is Gly-X-Y, with X often representing proline and Y hydroxyproline; these relative locations in the helix are essential for their contributions to its formation.⁴ Even working in H. polymorpha, which in addition to not being cultured in most labs has finicky codon preferences,⁵ there are a number of problems.

Fortunately for what was rapidly becoming an Escherichia coli-centric team, there exists a whole class of proteins known as "bacterial collagens". These proteins have the same supersecondary structure and Gly-X-Y repeat as human collagen without relying on hydroxyproline in the Y position,⁷ making these sequences easier to manipulate and bacterial synthesis possible. Unfortunately, despite the suggestive moniker, bacterial collagen has not been well adapted to provide structural integrity to large regions of extracellular matrix in areas of regular high stress such as arterial walls and skin. As such, it has no natural mechanism to form a network of long, cross-linked fibers, and there is a dearth of data on how such a matrix might perform. Still, it might prove a viable collagen mimetic given successful assembly of a fibrous matrix. Returning to human collagen mimetics for a moment, the length of native collagen has been one factor that makes production difficult, and there has been some interesting work done with relatively short peptides working around this difficulty.

The bacterially produced collagen mimetic we designed draws from a few additional sources, wading into the uncertain waters of rational design in the process. Yoshizumi et al. were able to demonstrate the utility of a designed coiled-coil homotrimerization domain in assisting the folding of a bacterial collagen domain.¹² Coiled-coil domains, the most famous example of which forms the fundamental dimer structure of keratin, are defined by a heptad repeat structure and can govern the association of anywhere between two and four proteins fairly easily;¹³ the example here was designed to form homotrimers which retained their structure above 90 °C.¹² The authors evaluate the effect of different locations of this domain on the refolding of a single bacterial collagen domain, concluding that a single coiled-coil domain at the N-terminus is optimal for the most rapid and complete refolding.¹² We created a close analog to this construct (modified for convenient PCR amplification) with a purification tag and submitted it as BBa_K2027007.

This general approach might be extended to different coiled-coil and target proteins; in particular, the potential applications of an effective heterotrimerization domain are even more expansive. A candidate coiled-coil obligate heterotrimer with a defined orientation was designed in 1995 by Nautiyal et al., with all homotrimers and two-component heterotrimers made unfavorable by charge pair interactions.¹³ Full heterotrimers have a higher melting point (apparent T_m = 87.5 °C) than the next-most stable arrangement by at least 15 °C,¹³ potentially allowing thermal cycling to increase specificity. A first step toward using this domain would be attempting to replace the coiled-coil in Yoshizumi et al.'s device with the coiled-coil domains making up the heterotrimer; bricks BBa_K2027044, BBa_K2027037, and BBa_K2027038 represent these constructs. Observing under what conditions, if any, specificity is maintained in the presence of a weaker homotrimerization domain (the bacterial collagen) could give some indication of the utility of these domains in organizing proteins. Collagen-like triple helices generally have melting temperatures below 40 °C, so the coiled-coil domains have a clear thermodynamic advantage and at least one has been shown to nucleate trimerization of an unfolded bacterial collagen trimer, but the kinetics of initial formation of trimers was not studied and in fact could not have been studied in the setting of the first construct. Instead, the bacterial collagen domain was unfolded while leaving the coiled-coil trimers intact;¹² it remains possible that at room temperature, interaction of bacterial collagen domains dominates the trimerization process. The relative stability of the heterotrimer will allow for formation of the desired heterotrimeric construct at an elevated temperature regardless of room temperature kinetics, but it would be interesting to study the kinetics nonetheless. Still, all constructs so far are short, blunt-ended fibers incapable of assembling into a matrix. However, the modular nature of this assembly suggests the possibility of large-scale cross-linking in a way peptide tesselation models might have more difficulty supporting.

Cross-linking is essential for proper functioning of collagen in a structural context; covalent linkages ensure not just thermal but also mechanical stability of quaternary protein associations. Bacterial collagen offers no help here; human collagen is replete with intra- and inter-strand cross-links but they depend on precise positioning and the action of multiple mammalian enzymes.⁶ Fortunately, a simple protein fiber cross-linking module has already had its efficacy demonstrated: exons 21 and 23 from human tropoelastin have been shown to cross-link coacervated elastin-like monomers in the presence of a lysine-oxidizing agent.¹⁴ The single relavant enzyme is not even necessary; as is shown in the PQQ aptamer purification project, it might soon be enough to purchase a cofactor supplement at a local drug store and add copper sulfate. To explore this, another set of constructs was created with this cross-linking domain at the N-terminus. These are the relevant bricks: BBa_K2027003, BBa_K2027004, BBa_K2027005, and BBa_K2027006. These constructs need to be studied to evaluate the effect of the addition on trimerization and the potential for cross-linking. Even if cross-linking is successful, these would still be the short, blunt fibers shown in Figure 2, but governance of their assembly by formation of a heterotrimer opens the door to sticky end use.

Parts BBa_K2027002 and BBa_K2027045 are the conjugations of the three heterotrimer parts (with and without cross-linking domains, respectively). Part BBa_K2027002 is represented in both detailed and simplified schematics in Figure 3. Due to the directionality and specificity of trimer formation, the preferred assembly has sticky ends leaving the possibility of substantially longer fibers such as that shown in simplified form in Figure 4.

We took the collagen constructs (mentioned in the previous section) and transformed them into E. coli. We picked successfully transformed colonies and ran colony polymerase chain reaction (CPCR) using the primers VF2 and VR. We ran these PCR products on agarose gels to examine whether the plasmids contained appropriately sized DNA. After successfully sequencing them to confirm that they indeed had the correct domain constructs, we wanted to induce the cells to produce the collagen proteins in order to characterize them. In addition, we wanted to also produce constructs without the elastin cross-linking domain. We removed this domain through Sap1 digestion of the DNA constructs and further transformed them into cells. We once again confirmed the success of the transformation with the digested constructs through gel electrophoresis and sequence verification. Although our sequence verifications were impressively similar to our desired constructs, it actually took 3 digestion attempts to fully digest the constructs to produce cells with constructs BBa_K2027044, BBa_K2027037, and BBa_K2027038. While it is successful, the number of tries suggests that our Sap1 digestion with Golden Gate Assembly protocol could definitely be improved to increase efficiency.

After induction of our liquid cultures, we extracted the proteins from our constructs. We kept all protein washes to assure that our desired protein product did not get extracted earlier than expected (we expected the protein to be in the final elution buffer washes). After confirming its presence in the final elution washes (shown in Figure 6), we ran the extracts against each other. To verify the size of the protein, we ran our protein extractions in SDS-PAGE gels. We mostly used Ni columns to purify our protein extract. Since our construct has a Lumio-Flag-His tag, we also used Anti-FLAG magnetic beads, with the help of our mentor Kosuke, to examine which protocol was most efficient in extracting protein.

Figure 8 also represents an SDS-PAGE gel containing the protein extracts of BBa_K2027003 (natural construct without the elastin cross-linking domain or SN) in lanes 2-4 and BBa_K2027002 in lanes 5-7. BBa_K2027002 is the fully assembled heterotrimeric coiled-coil collagen fiber monomer, consisting of the domains BBa_K2027004, BBa_K2027005, and BBa_K2027006. Because the fully assembled construct contains three domains, we expected the protein produced would be at least three times larger than the proteins with singular constructs. However, as indicated by the gel in Figure 2, BBa_K2027002 did not produce this size, which suggested that the assembly of all parts may have introduced errors in the protein synthesis.

We also explored the possibility of cross-linking the proteins. Our plan was to cross-link the proteins and compare the results with un-processed protein in an SDS-PAGE. Initially, we wanted to test small samples of cross-linked C1, C2, and C3 and S1, S2, and S3 against non-cross-linked samples. We expected cross-linked proteins to have a higher molecular weight than proteins that weren't. Since this was our first time attempting to cross-link proteins, we wanted to test our protocol on only one sample before proceeding on a large-scale cross-linking experiment.

In this experiment, we used pure pyrroloquinoline quinone (PQQ) as a catalyst for the cross-linking reaction for sample construct CN. PQQ was diluted in a copper (II) solution and mixed with the purified protein. Since the purified protein exists in a phosphate buffer (extract was purified from the Ni-NTA resin columns), we maintained the pH of the reaction solution and kept it close to pH 7 while adding excess copper because the precipitation of copper(II) phosphate causes an increase in acidity of the buffer. When the solution became basic, we found that the solution was mostly clear and the precipitate was a light green-blue color, likely copper hydroxide. When the solution was nearly pH 7 and borderline acidic, the solution turned a clear blue and produced a fine blue copper(II) phosphate precipitate, but copper remained in solution. We incubated the two different sample solutions at room temperature for 24 hours. After 24 hours, we found that the solutions separated into two layers. We weren't sure if the cross-linked protein would exist in the precipitate-like layer or the supernatant so we ran both against the original untreated CN in a gel.