Difference between revisions of "Part:BBa K3089006"
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<h3>Introduction</h3>
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<h3> Introduction </h3>
 
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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 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.
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  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.
 
  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.
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<h3> Protein expression </h3>
 
<h3> Protein expression </h3>
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<figcaption> The circuit of the protein BBa_K30889026 </figcaption>
 
  
 
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The predicted size of rBalcp19k-linker-mfp5 is 28.17 kDa, and the isoelectric point is 10.41. rBalcp19k-linker-mfp5 was cloned into pET28b and expressed in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃.  
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<i>mTyr-CNK</i> with tag for purification was cloned into pET28b and expressed in <i>E.coli</i> 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 <b>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.</b>
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In order to detect its expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that no protein bands of rBalcp19k-linker-mfp5(~28 kDa) could be observed on lane rBalcp19k-mfp5 compared with lane pET28b (pET28b empty vector)(Figure 1A), which means the expression of this protein is not well in BL21(DE3) Rosetta.  
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We can get the same results using quantitative densitometry analysis of SDS-PAGE gels (Figure 1B).
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<figcaption> Figure 2 Detection of the expression level of all recombinant proteins by SDS-PAGE.(A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots. </figcaption>
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<figcaption> Figure 2. Purification of mTyr-CNK and test of <i>in vitro</i> tyrosine hydroxylation by NBT staining. (A) Cell pellets collected after protein expression. NC: empty vector; mTyr-CNK: tyrosinase from marine microorganism; (B) SDS-PAGE of purified mTyr-CNK by affinity chromatography.  
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<h3> Protein purification </h3>
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<h3> <i>In vitro</i> DOPA modification and NBT staining </h3>
  
 
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Barnacle cement proteins are very promising in making biomedical bio-glues. rBalcp19K from Balanus albicostatus had the properties of both self-assembly and adhesion.
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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 (<u>see details on our methods</u>). 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 <i>in vitro</i> modified recombinant protein performed positive result in NBT staining test, <b>which showed tyrosines of these proteins were modified into DOPA by mTyr-CNK</b>, BSA protein was used as negative control (Figure 3).
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It also could function in more basic condition than Mfps. Thus we also designed a novel recombinant protein by combining it with Mfp5. We expected rBalcp19k-Mfp5 would perform better adhesive ability to solidify our idea of modularisation of Mfp5.  
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We tried to purify it under native conditions, and we found bands of rBalcp19K-linker-mfp5 appeared between 25kDa and 35kDa on 12% SDS-PAGE gel(Figure 2), which meant it was successfully expressed and purified under native condition(see details on our methods).  
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Protein concentrations of rBalcp19k-linker-mfp5 were measured by BCA assay, and its yield is 1mg/L.
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Revision as of 14:44, 19 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).

Figure 1. Catalytic activity of tyrosinase and advantages of mTyr-CNK.

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
  • In vivo DOPA modification by co-expression

    • 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.

    Figure 2. Purification of mTyr-CNK and test of in vitro tyrosine hydroxylation by NBT staining. (A) Cell pellets collected after protein expression. NC: empty vector; mTyr-CNK: tyrosinase from marine microorganism; (B) SDS-PAGE of purified mTyr-CNK by affinity chromatography.

    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).

    Figure 3. SDS-PAGE of purified rBalcp19k-mfp5 by affinity chromatography under native conditions. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant protein rBalcp19K; S, soluble cell fraction; W1, fraction.

    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.

    Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).