Parts

Introduction

The overarching aim of the Renervate Therapeutics project is to develop a synthetic, mussel-based protein polymer to coat a 3D-printed polycaprolactone (PCL) scaffold, which would allow axonal regeneration in spinal cord injury. Our mussel-based protein polymer is derived from PVFP-5, a mussel foot protein (MFP) secreted by Perna viridis. PVFP-5 has well documented non-cytotoxic properties, and possesses strong adhesive properties due to its natural role in attaching to underwater substrates (Santonocito et al., 2019). Our PCL scaffold is not adhesive, therefore PVFP-5 is a good candidate to allow adherence of our PCL scaffold in the spinal micro-environment.

Renervate Therapeutics utilised three basic parts this year; PVFP-5, a mussel foot protein (MFP) secreted by Perna viridis, the tyrosinase enzyme from Bacillus megaterium, and a thermostabilised variant of the Chondroitinase ABC enzyme (ChABC) from Proteus vulgaris. ChABC is an enzyme that degrades chondroitin sulfate proteoglycans (CSPG) and has the potential to improve axonal sprouting following SCI. Three composite parts were used; PVFP-5 (BBa_K3794005), Tyrosinase (BBa_K3794003) and Tyrosinase (BBa_K3794005) coding sequences with a Lac regulatory system, and a rrnB1 T1 Terminator (for ChABC and Tyrosianse only). Each part was cloned into a high-copy number plasmid - pSB1A3 - for subsequent expression in various E.coli cell lines; including BL21, BL21 (DE3), Rosetta-gami B (DE3), and SHuffle (DE3).

Table 1: A list of our basic and composite parts.

Part Name	Part Number	Description	Type	Length (bp)
PVFP-5 CDS	BBa_K3794000	PVFP-5 insert with stop codon and N-terminal 6xHis tag+TEV site.	Basic	417
Tyrosinase CDS	BBa_K3794002	Tyrosinase insert with stop codon.	Basic	897
ChABC CDS	BBa_K3794004	ChABC insert with stop codon and N-terminal 6xHis tag+TEV site.	Basic	3042
Lac Regulator	BBa_R0010	Lac Promoter encoding CAP Binding site, Lac promoter and Lac operator. Sensitive to CAP and LacI	Regulatory	200
Terminator	BBa_B0010	rrnB T1 Terminator	Terminator	80
PVFP-5 Composite Part	BBa_K3794001	Translational unit for PVFP-5. Encodes Lac regulatory system, as well as PVFP-5 with upstream 6xHis Tag and TEV cleavage site	Composite	617
Tyrosinase Composite Part	BBa_K3794003	Translational unit for Tyrosinase encoding Lac regulatory system, Tyrosinase coding sequence and rrnB1 T1 Terminator.	Composite	1177
ChABC Composite Part	BBa_K3794005	Translational unit for ChABC encoding Lac regulatory system, ChABC coding sequence with upstream 6xHis tag and TEV cleavage site. rrnB T1 Terminator found on 3’ end.	Composite	3322

Basic Parts

PVFP-5 (BBa_K3794000)

PVFP-5 is an adhesive protein secreted by the Asian mussel Perna viridis. It is expressed by the foot of the byssal thread, at the interface between the filament and the target surface. Here, it uses its high L-3,4-dihydroxyphenylalanine (L-DOPA) content to help the mussel stick to a wide range of non-specific surfaces (Santonocito et al., 2019). As a result of the COVID-19 pandemic, our ongoing collaboration with Prof Annalisa Pastore was no longer logistically feasible. Therefore, we re-designed our bioadhesive protein of choice from PVFP-5β to PVFP-5.

Our PVFP-5 construct was designed using the sequence derived from UniProtKB U5Y3S6 (Guerette et al., 2013), and we removed the signal peptide (residues 1-18). We codon optimised for E.coli expression and synthesised the construct through IDT, with an N-Terminal 6xHis tag and TEV cleavage site. Upstream of these tags, is a start codon to allow utilisation of this basic part for both protein expression and purification.

Figure 1: PVFP-5 Basic part (BBa_K3794000). 6xHis tag+TEV cleavage site (pink) and PVFP-5 coding sequence (purple) are labelled. Figure developed from SnapGene.

Tyrosinase (BBa_K3794002)

The Tyrosinase enzyme from Bacillus megaterium is a copper-containing oxidoreductase enzyme that acts on the phenol groups of tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) (Shuster Ben-Yosef, Sendovski and Fishman, 2010). The BBa_K794002 sequence was obtained from the iGEM Registry from Part BBa_K3033013, and subsequently codon optimised for expression in E.coli. As the use of tyrosinase in our project is limited to in vivo post-translational modification of tyrosine to L-DOPA on the PVFP-5 derived from BBa_K3794001, there has been no addition of a 6xHis tag or TEV cleavage site as protein purification will not be required.

Figure 2: Tyrosinase Basic part (BBa_K3794002). The Tyrosinase coding sequence (purple) is labelled. Figure developed from SnapGene.

ChABC (BBa_K3794004)

This year we employed the use of ChABC, a bacterial enzyme which functions to degrade chondroitin sulfate proteoglycans (CSPGs) and is key for overcoming the effects caused by CSPGs, in the microenvironment of SCI, to inhibit endogenous regeneration. The amino acid sequence for our ChABC enzyme was derived from UniProtKB P59807 (Huang et al., 2003). We then optimised the nucleotide sequence for expression in E.coli. ChABC from Proteus vulgaris has well documented thermal instability at 37°C, therefore we introduced a series of eight point mutations in this protein to confer thermostability at this temperature (Hettiaratchi et al., 2020). Further information regarding these mutations can be found here

ChABC was synthesised through IDT with an N-Terminal 6xHis tag and TEV cleavage site, upstream of these tags is a start codon to allow this basic part to be used for protein expression and protein purification.

Figure 3: ChABC Basic part (BBa_K3794004). TEV cleavage site (pink) and ChABC coding sequence (purple) are labelled. Figure developed from SnapGene.

Plasmid Backbone

pSB1A3

The pSB1A3 plasmid is the main plasmid backbone utilised by Renervate Therapeutics for the expression of our three recombinant proteins. pSB1A3 is an ampicillin resistant, high-copy number plasmid (100-300) and contains a pUC19-derived pMB1 origin of replication. The BioBrick prefix and suffix are flanked by two bacterial promoters which prevent transcription from inside the multiple cloning site (MCS) from reading out into the plasmid.

Figure 4: pSB1A4 plasmid backbone, including EcoRI, XbaI, SpeI, PstI restriction enzymes. Figure developed from SnapGene.

Composite Parts

PVFP-5 Composite Part (BBa_K3794001)

The PVFP-5 composite part contains BBa_R0010, an inverting regulator sensitive to CAP and LacI, alongside the PVFP-5 basic part (BBa_K3794000) to form the translational unit for PVFP-5 expression in E.coli. In our project, this composite part was ligated into pSB1A3, therefore the plasmid bacterial terminator was relied upon for translational termination. This composite part was synthesised by IDT.

Figure 5: PVFP-5 Composite part (BBa_K3794001). 6xHis tag+TEV cleavage site (pink), CAP binding site and lac operon (teal), lac promoter (white arrow), BBa_R0010 contains all the elements in teal and the lac promoter, Biobrick prefix and Biobrick suffix (light blue) and PVFP-5 coding sequence (purple) are labelled. Figure developed from SnapGene.

Tyrosinase Composite Part (BBa_K3794003)

The Tyrosinase composite part contains BBa_R0010, an inverting Lac regulator sensitive to CAP and LaI, as well as the basic part for Tyrosinase (BBa_K3794002) to form the translational unit for Tyrosinase in E.coli. This composite part also contained a rrnB T1 Terminator (BBa_B0010). This composite part was synthesised by IDT.

Figure 6: Tyrosinase Composite part (BBa_K3794003). CAP binding site and lac operon (teal), lac promoter (white arrow), BBa_R0010 contains all the elements in teal and the lac promoter, Biobrick prefix and Biobrick suffix (light blue) and Tyrosinase coding sequence (purple) are labelled. Figure developed from SnapGene.

ChABC Composite Part (BBa_K3794005)

The ChABC composite part contains BBa_R0010, an inverting Lac regulator sensitive to CAP and LacI, as well as the basic part for ChABC (BBa_K3794004) to form the translational unit for ChABC expression in E.coli. This composite part also contains a rrnB T1 Terminator (BBa_B0010) for translational termination. This composite part was synthesised by IDT.

Figure 7: ChABC Composite part (BBa_K3794005). TEV cleavage site (pink), CAP binding site and lac operon (teal), lac promoter (white arrow), BBa_R0010 contains all the elements in teal and the lac promoter, Biobrick prefix and Biobrick suffix (light blue) and ChABC coding sequence (purple) are labelled. Figure developed from SnapGene.

References:

Guerette, P. A., Hoon, S., Seow, Y., Raida, M., Masic, A., Wong, F. T. et al. (2013). Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science. Nature biotechnology, 31(10), 908-915.
Hettiaratchi, M., O’Meara, M., O’Meara, T., Pickering, A., Letko-Khait, N., & Shoichet, M. (2020). Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability. Science Advances, 6(34), eabc6378. doi: 10.1126/sciadv.abc6378
Huang, W., Lunin, V., Li, Y., Suzuki, S., Sugiura, N., Miyazono, H., & Cygler, M. (2003). Crystal Structure of Proteus vulgaris Chondroitin Sulfate ABC Lyase I at 1.9Å Resolution. Journal of Molecular Biology, 328(3), 623-634. doi:10.1016/s0022-2836(03)00345-0
Santonocito, R., Venturella, F., Dal Piaz, F., Morando, M. A., Provenzano, A., Rao, E. et al. (2019). Recombinant mussel protein Pvfp-5β: A potential tissue bioadhesive. Journal of Biological Chemistry, 294(34), 12826-12835. doi:10.1074/jbc.ra119.009531

Team:KCL UK/Parts