Difference between revisions of "Team:Shenzhen SFLS/Protein Engineering"
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<a href="https://2015.igem.org/Team:Shenzhen_SFLS/Protein_Engineering_M1">Next</a> | <a href="https://2015.igem.org/Team:Shenzhen_SFLS/Protein_Engineering_M1">Next</a> | ||
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<p><h2><b>3.1 Protein engineering</h2></b></p> | <p><h2><b>3.1 Protein engineering</h2></b></p> | ||
<p><blockquote> P185<sup>her2/neu</sup>, member of the HER receptor family, is chosen as the antigen to be mutated in our project. We used several programs to help us have a better overall understanding of the antigen. Since only the extracellular domain can be recognized and bound by an antibody, we would only mutate and express the extracellular domain (ECD). Also, some results below only cover ECD and its mutants.</blockquote></p> | <p><blockquote> P185<sup>her2/neu</sup>, member of the HER receptor family, is chosen as the antigen to be mutated in our project. We used several programs to help us have a better overall understanding of the antigen. Since only the extracellular domain can be recognized and bound by an antibody, we would only mutate and express the extracellular domain (ECD). Also, some results below only cover ECD and its mutants.</blockquote></p> | ||
| − | <center><a href="https://static.igem.org/mediawiki/2015/9/90/PRdePE_M0_result.txt"> | + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/d/d2/PRdePE_M0_polarity.gif | + | <center><a href="https://static.igem.org/mediawiki/2015/9/90/PRdePE_M0_result.txt">Numerical_result_of_p185<sup>her2/neu</sup> ECD_M0</a></center> |
| − | </br><i><center>Fig 3.1 .Polarity scale of | + | |
| − | <center><img src="https://static.igem.org/mediawiki/2015/c/cd/PRdePE_M0_HDRPH.gif | + | <center><img src="https://static.igem.org/mediawiki/2015/d/d2/PRdePE_M0_polarity.gif"/></center> |
| − | </br><i><center>Fig 3.2 .Hydrophobicity scale of | + | |
| + | </br><i><center>Fig 3.1 .Polarity scale of p185<sup>her2/neu</sup> ECD, predicted by ProtScale, Zimmerman polarity</br>Window size: 9. Scale not normalized. Relative weight for window edges: 100 %. Weight variation model: linear.(7)</center></i> | ||
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| + | <center><img src="https://static.igem.org/mediawiki/2015/c/cd/PRdePE_M0_HDRPH.gif" /></center> | ||
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| + | </br><i><center>Fig 3.2 .Hydrophobicity scale of p185<sup>her2/neu</sup> ECD, predicted by ProtScale, Hphob. / Eisenberg et al.</i></center> | ||
</br> | </br> | ||
| − | <center><img src="https://static.igem.org/mediawiki/2015/3/3e/PRdePE_M0_2ND_structure.png | + | |
| − | </br><i><center><blockquote><blockquote><blockquote> | + | <center><img src="https://static.igem.org/mediawiki/2015/3/3e/PRdePE_M0_2ND_structure.png"/></center> |
| + | </br><i><center><blockquote><blockquote><blockquote><blockquote><blockquote><blockquote>Fig 3.3. Secondary structure and solvent accessibility prediction of p185<sup>her2/neu</sup> ECD, predicted by PredictProtein. The black scale plate above with numbers indicates the number of residues. Corresponding to the residue numbers, the stripes below the scale plate indicate the secondary structure of the region covered. Blue stripes indicate strand region, while red stripes indicate helix region. Results of the first and second row are respectively predicted by RePROF (new and experimental) and PROFsec (veteran). Results of the third row indicate whether the corresponding region is exposed or buried. Blue stripes indicate that the regions covered are exposed, and yellow stripes indicate that the regions covered are buried.(7)</blockquote></blockquote></blockquote></blockquote></blockquote></blockquote></center></i> | ||
</br> | </br> | ||
| − | <center><img src="https://static.igem.org/mediawiki/2015/d/d2/PRdePE_M0_3rd_structure.png | + | |
| − | </br><i><center> | + | <p><blockquote>After further research, we chose antibody chA21 as the antibody in our project. According to the research of Zhou H. et al, we were able to learn the key residues of each loop of p185<sup>her2/neu</sup>.</p></blockquote> |
| − | <center><img src="https://static.igem.org/mediawiki/2015/5/59/PRdePE_M0_local_quality_estimate.png | + | |
| − | </br><i><center> | + | <center><img src="https://static.igem.org/mediawiki/2015/e/e1/PRdePE_interface.PNG" /></center> |
| + | </br> | ||
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| + | </br><i><center><blockquote><blockquote><blockquote><blockquote><blockquote><blockquote>Fig 3.4. The scFv-EPI interface. EPI consists of the p185<sup>her2/neu</sup> ECD subdomain I (residues 1~172) and the first C2 module (residues 173~192) of subdomain II, which are respectively colored in green and yellow. Only the three loops containing the epitope are drawn. The light chain (VL) and heavy chain (VH) are colored in blue and red respectively. Other parts of scFv chA21 are colored in grey. A. The stereo overview of the scFv-EPI interface. B. The three loops containing the epitope of EPI lie on the chA21 surface. The important residues on EPI are shown as sticks and labeled with black characters. C-F, the detailed hydrogen bonding interaction network between chA21 and EPI. The hydrogen bonds are drawn as grey dashes. The residues are labeled, and characters in the parentheses indicate which CDR (chA21 scFv) or loop (EPI) the residues belong to. In panels B-F, the oxygen, nitrogen, and sulfur atoms are colored red , blue and orange, respectively.(9)</blockquote></blockquote></blockquote></blockquote></blockquote></blockquote></br></i></center> | ||
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| + | <p><blockquote>We used SWISS MODEL to predict the tertiary structure of p185<sup>her2/neu</sup> ECD. </p></blockquote> | ||
| + | |||
| + | <center><img src="https://static.igem.org/mediawiki/2015/d/d2/PRdePE_M0_3rd_structure.png" /></center> | ||
| + | </br> | ||
| + | |||
| + | </br><i><center>Fig 3.5. Tertiary structure of p185<sup>her2/neu</sup> ECD, predicted by SWISS MODEL.(9)</i></center> | ||
| + | |||
| + | <center><img src="https://static.igem.org/mediawiki/2015/5/59/PRdePE_M0_local_quality_estimate.png" /></center> | ||
| + | </br><i><center>Fig3.6. Local quality estimate of p185<sup>her2/neu</sup> ECD, predicted by SWISS MODEL.(9)</center></i> | ||
| + | <p><blockquote>From the figure above and other supplemental materials according to the research of Zhou H. et al, we were able to make sure which residues we were going to mutate. </p></blockquote> | ||
| + | |||
| + | <p><blockquote>We used several approaches to predict the parameters and structures of the mutants of p185her2/neu ECD. Please click the "Next" below to get detailed information.</blockquote> | ||
</p> | </p> | ||
<p id="invest"> | <p id="invest"> | ||
Latest revision as of 03:27, 19 September 2015
There are 9 pages
3.1 Protein engineering
P185her2/neu, member of the HER receptor family, is chosen as the antigen to be mutated in our project. We used several programs to help us have a better overall understanding of the antigen. Since only the extracellular domain can be recognized and bound by an antibody, we would only mutate and express the extracellular domain (ECD). Also, some results below only cover ECD and its mutants.
Fig 3.3. Secondary structure and solvent accessibility prediction of p185her2/neu ECD, predicted by PredictProtein. The black scale plate above with numbers indicates the number of residues. Corresponding to the residue numbers, the stripes below the scale plate indicate the secondary structure of the region covered. Blue stripes indicate strand region, while red stripes indicate helix region. Results of the first and second row are respectively predicted by RePROF (new and experimental) and PROFsec (veteran). Results of the third row indicate whether the corresponding region is exposed or buried. Blue stripes indicate that the regions covered are exposed, and yellow stripes indicate that the regions covered are buried.(7)
After further research, we chose antibody chA21 as the antibody in our project. According to the research of Zhou H. et al, we were able to learn the key residues of each loop of p185her2/neu.
Fig 3.4. The scFv-EPI interface. EPI consists of the p185her2/neu ECD subdomain I (residues 1~172) and the first C2 module (residues 173~192) of subdomain II, which are respectively colored in green and yellow. Only the three loops containing the epitope are drawn. The light chain (VL) and heavy chain (VH) are colored in blue and red respectively. Other parts of scFv chA21 are colored in grey. A. The stereo overview of the scFv-EPI interface. B. The three loops containing the epitope of EPI lie on the chA21 surface. The important residues on EPI are shown as sticks and labeled with black characters. C-F, the detailed hydrogen bonding interaction network between chA21 and EPI. The hydrogen bonds are drawn as grey dashes. The residues are labeled, and characters in the parentheses indicate which CDR (chA21 scFv) or loop (EPI) the residues belong to. In panels B-F, the oxygen, nitrogen, and sulfur atoms are colored red , blue and orange, respectively.(9)
We used SWISS MODEL to predict the tertiary structure of p185her2/neu ECD.
From the figure above and other supplemental materials according to the research of Zhou H. et al, we were able to make sure which residues we were going to mutate.
We used several approaches to predict the parameters and structures of the mutants of p185her2/neu ECD. Please click the "Next" below to get detailed information.
There are 9 pages