Team:CAU China/Modeling

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Pre analysis

Before we started to do our in-lab experiments, we did some analysis of 2A system to help us build a better device. According to previous reports and our analysis, these factors will influence the efficiency of 2A cleavage: (i) 2A sequence (ii) upstream sequences of 2A (iii) The length of 2A.

As for the sequence of 2A, we analyze the sequence of several natural different 2A linkers. The result shows below.

So there are several conserved residues in 2A sequence, site mutant experiment carried by Ryan shows that mutant of these conserved residues may lead to much lower cleavage efficiency.

In order to know why upstream sequence and length of 2A will influence cleavage efficiency, we firstly have to take a deep look at the mechanism of 2A cleavage.

The most acceptable model shows 2A mediates a cotranslational “ribosome skipping” event here, it predicts that the nascent 2A interacts with the exit tunnel of the ribosome to affect the conformational space occupied by the ester bond linking the nascent protein and tRNAGly in the ribosome P-site. The model predicts that the residues may influence activity reside within the exit tunnel of the ribosome. Also the structural data shows that the ribosome exit tunnel may accommodate 30-40 amino acids. So the activity of shorter forms of F2A may be affected by the nature of the C-terminal sequence of the protein upstream .Then we use secondary structural prediction program to predict the 2A region structure, the result showed that it will form an a helices capped that their C termini. And this has been more stringently tested by dynamic molecular modeling studies on FMDV 2A by J.Wilkie.

Prediction result from Protein Homology/analogY Recognition Engine V 2.0.

To get the most suitable length of 2A, we analyze the stability of 2A linkers with different lengths. Here we use ProtParam to analyze the 2A sequence listed on the paper to calculate the unstable index. The instability index provides an estimate of the stability of your protein in a test tube. Statistical analysis of 12 unstable and 32 stable proteins has revealed [7] that there are certain dipeptides, the occurrence of which is significantly different in the unstable proteins compared with those in the stable ones. The authors of this method have assigned a weight value of instability to each of the 400 different dipeptides (DIWV). Using these weight values it is possible to compute an instability index (II) which is defined as:

DIWV(x[i]x[i+1]) is the instability weight value for the dipeptide starting in position i.

A protein whose instability index is smaller than 40 is predicted as stable, a value above 40 predicts that the protein may be unstable. The statistical result shows here.

Since 2A with 22 amino acids shows lowest unstable index, it is the most stable one, which will unlikely to be affected by upstream sequences.

So using the results of our pre analysis, we constructed our device and did our experiments.

Reference

[1]Claire Halpin,Susan E. Cooke, Abdellah Barakate,Abdel El Amrani and Martin D. Ryan.Self-processing 2A-polyproteins – a system for co-ordinate expression of multiple proteins in transgenic plants.The Plant Journal (1999) 17(4), 453–459.

[2]Ekterina Minskaia and Martin D.Ryan.Protein Coexpression Using FMDV 2A Effect of "Linker" Residues.BioMed Research International 2013;Article ID 291730

[3]Michelle L. L. Donnelly, Garry Luke,Amit Mehrotra,Xuejun Li,Lorraine E. Hughes,David Gani and Martin D. Ryan.Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’mechanism indicates not a proteolytic reaction, but a novel translational effect : a putative ribosomal ‘skip’.Journal of General Virology (2001), 82, 1013–1025

[4]Ekaterina Minskaia, John Nicholson and Martin D Ryan.Optimisation of the foot-and-mouth disease virus 2A co-expression system for biomedical applications.BMC Biotechnology 2013, 13:67

[5]Martin D. Ryan, Michelle Donnelly, Arwel Lewis, Amit P. Mehrotra,John Wilkie, and David Gani.A Model for Nonstoichiometric, Cotranslational Protein Scission in Eukaryotic Ribosomes.Bioorganic Chemistry 27, 55–79 (1999)

[6]Pablo de Felipe, Garry A. Luke, Jeremy D. Brown and Martin D. Ryan.Inhibition of 2A-mediated ‘cleavage’ of certain artificial polyproteins bearing N-terminal signal sequences.Biotechnol. J. 2010, 5, 213–223