Difference between revisions of "Part:BBa K3794001"

Revision as of 21:52, 21 October 2021

PVFP-5 Composite

This composite part is a translational unit for PVFP-5. It is used for expression of a 6xHis tagged PVFP-5 protein, BBa_K3794000, in E.coli. It is under the control of the lac promoter and operator. It also contains a CAP binding site. It has been codon optimised for expression in E.coli K12, and was used with pSB1A3 by KCL iGEM 2021.

Expression can be conducted using IPTG-induction, and the expressed protein can be purified using a Ni-NTA column.

This composite part / translational unit does not contain a terminator, therefore a plasmid, such as pSB1A3, containing a terminator region or an addition terminator part from the iGEM registry is required.

Introduction

PVFP-5 is a mussel foot protein secreted by the Asian green mussel species, Perna viridis . PVFP-5 has been investigated widely for uses in biomedical applications recently, due to its ability to retain strong adhesive properties in aqueous conditions, as well as exhibiting biocompatibility and non-cytotoxicity within in vivo experiments (Santonocito et al., 2019). This makes it ideal for applications in medicine, biotechnology, and material science. The adhesive properties of PVFP-5 have been attributed to the post-translationally modified version of tyrosine, L-DOPA. PVFP-5 has been found to consist of up to 21% L-DOPA residues, making it the most adhesive mussel foot protein secreted by Perna viridis and hence likely to function as a successful bioadhesive.

Structural Modelling

After sequence and domain analysis of PVFP-5 (U5Y3S6), we predicted that it will form 9 disulphide bonds (3 in each domain of PVFP-5). We produced a full structural modelling using a combination of homology modelling and molecular dynamics (Figure 1).

Having learnt that our protein structure has a high disulphide bond content, we designed our expression system to include E.coli cell lines that are optimised for disulphide bond formation - to allow correct folding and synthesis of our protein in its soluble form.

DNA Amplification and DNA Purification

PVFP-5 Composite, BBa_K3794001, a 617bp long DNA construct was amplified using PCR and a thermal cycler. Promega GoTaq Green Master Mix was used for PCR. PCR products from BBa_K3794001 were run alongside BBa_K3794003 on a 2% Agarose gel. The PCR products for BBa_K3794001 and BBa_K3794003 can be seen in Figure 2. PVFP-5 / BBa_K3794001 is 617bp long, and correlates with the 600bp band on the 100bp ladder, and therefore we can infer that our PCR was successful.

Expression and Purification

BBa_K3794001 was expressed initially in two E.coli cell lines; BL21 (DE3) and Rosetta-Gami B (DE3) - the latter being optimised for disulphide bond formation. BL21 (DE3) was used as a control against Rosetta-Gami B to measure which cell line is most optimal.

Expression was induced with IPTG at 37C, and samples at intervals were taken to visualise the progression of expression. An SDS-PAGE of our protein expression can be seen below.

As seen in Figure 3, around the 15kDa band for lanes 4 and 5, and 8 and 9, we have suspected strong expression of PVFP-5 from BBa_K3794001. To confirm this, we had to purify our E.coli samples.

Protein purification was conducted after expression. Ni-NTA spin column purification was utilised - SDS-PAGE results showed a very low yield of eluted protein after purification. Unfortunately, an SDS-PAGE gel of this is not available.

Expression and Purification 2.0

Following our low yield of purified protein, yet having suspected strong expression (Figure 3), we reevaluated our expression and purification methodology. We re-expressed BBa_K3794001 using E.coli SHuffle (DE3) - another cell line optimised for disulphide bond formation, in the hope it would increase the soluble concentration of the protein. We conducted an SDS-PAGE analysis after this new expression which can be seen in Figure 4. In this expression system, PVFP-5 expressed by BBa_K3794001 was largely found in the insoluble form - in the total cell lysate (around 15kDa region, lane 2, 4, 6, and 8).

Suspecting that while we have expression of PVFP-5, yet cannot purify it to a sufficient yield, it may be due to the intrinsic adhesive ability of the protein. We hypothesised that PVFP-5 may be adhering to E.coli proteins and the E.coli membranes, and therefore cannot be easily isolated during protein purification.

To combat this, we introduced the use of 1% Triton X-100 in our lysis buffer to dissociate PVFP-5 from the cell-lysate. After protein purification via a Ni-NTA resin, we ran an SDS-PAGE gel to analyse the results from our purification. This can be seen in Figure 5

The yield of our purified / eluted protein remained quite low as can be seen in lanes 7 - 9, around the 15kDa band, despite using 1% Triton X-100 during our lysis.