Difference between revisions of "Part:BBa K3089006"

Revision as of 11:50, 20 October 2019

Introduction

This part is designed for mTyr-CNK protein purification and in vitro/vivo DOPA modification of all Mfp containing recombinant proteins. The unnatural amino acid DOPA plays important role in Mfp’s adhesion. In natural Mfps came from posttranslational modification(PTM) in mussel foot cell, but E coli was unable to do so. Tyrosinases could oxidize tyrosine to turn it into DOPA. Although there is an existing tyrosinase in the part registry already, our mTyr-CNK has a higher efficiency in catalyzing relevant reactions, thus is more suitable for DOPA modification to obtain a stronger adhesion. Instead of obtaining tyrosinase from Streptomyces sp. like in most studies, ours is found in a marine archaeon Candidatus Nitropumilus koreensis. This part was designed based on paper “A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase”(Do, Kang, Yang, Cha, & Choi, 2017).

Characterization

mTyr-CNK is well-expressed in E. coli during our experiments, and it successfully modified all of our recombinant proteins based on Mfp5 in vitro; the co-expression system where they are expressed together inside a cell for a more efficient in vivo modification also has a very interesting potential. We believe mTyr-CNK will become a very useful part for projects that involve DOPA modification. We also have various qualitative and quantitative data from protein expression, purification, and in vivo/ vitro DOPA modification. BBa_K3089006 was characterized in following experiments:

• protein expression and purification
• In vitro DOPA modification and NTB staining
• Surface coating analysis

Protein expression

mTyr-CNK with tag for purification was cloned into pET28b and expressed in E.coli BL21(DE3) by 500μM IPTG for 20h at 37℃.Interestingly, after expression, the sediment of bacteria showed rose colour(Figure 2A)，probably caused by the interference of mTyr-CNK in pigment pathway of E.coli BL21(DE3) Rosetta. The exact mechanism was remained unknown since the lack of research on this tyrosinase. Results showed that obvious protein bands of mTyr-CNK(~35 kDa) could be observed on lane WC compared with lane NC (pET28b empty vector)(Figure 1B), which means expression of this protein is well in BL21(DE3). Next We tried to purify mTyr-CNK under native conditions, and we found bands of mTyr-CNK appeared around 35kDa on 12% SDS-PAGE gel (Figure 2B), which meant it was successfully expressed and purified under native condition. Protein concentrations were measured by BCA assay and its yield is 7mg/L. Its yield is higher than any recombinant protein in our toolbox.

In vitro DOPA modification and NBT staining

In order to detect its modification ability on Mfp5 containing recombinant proteins, 10 ul 0.35mg/ml mTyr-CNK(in PBS, 0.02mM CuSO4）was added into 90ul protein solution of concentration 0.5m/ml（pH=6.0 PBS）for 3 hours in room temperature. Results were verified by NBT staining (see details on our methods). Dopa-containing proteins can be specifically stained by nitroblue tetrazolium (NBT) and glycinate solutions because they can catalyze redox-cycling reactions at an alkaline pH9. The NBT assay was thus used to confirm the successful post-translational modification of tyrosine into Dopa in modified proteins. All in vitro modified recombinant protein performed positive result in NBT staining test, which showed tyrosines of these proteins were modified into DOPA by mTyr-CNK, BSA protein was used as negative control (Figure 3).

Surface coating analysis

After obtaining a small number of recombinant proteins, surface coating analysis for qualitatively assessing the surface adsorption ability of recombinant proteins was conducted on two of most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates. As shown in Figure3, rBalcp19k-linker-mfp5 recombinant protein showed higher surface absorption abilities on both different substrates than rBalcp19k without fusion of mfp5 on its C-terminal. It’s suggested that Mfp improves the coating ability of rBalcp19k-linker-mfp5 fusion proteins. The In-vitro DOPA modification by mTyr-CNK tyrosinase significantly improved its surface absorption abilities, which suggested the positive contribution of DOPA in adhesive protein performances.

As shown in Figure 5, Mfp5 related proteins (unmodified) exhibited higher surface absorption abilities than other recombinant proteins,whereas almost all absorbed BSA were washed away. This revealed the functional advantage of Mfp5, since we could combine it with different proteins and predict the properties of recombinant proteins, making them more adaptable to diverse conditions. Furthermore, the DOPA modification by mTyr-CNK significantly improved the surface absorption abilities of Mfp-related recombinant proteins, which suggested the positive contribution of DOPA in adhesive protein performances. Future research on mechanism of DOPA may suggest more applications of DOPA, it is possible to even integrate DOPA into other proteins like fibrin to enhance them.