Expression optimization library of an artificial two domain cytokine.

<p>(A) Cytokine expression optimization library design. Listed functional modules were included in two separate reactions. In one reaction domain A is used at the N-TAG position and domain B at the core protein position. In the second reaction domain B is used at the N-TAG position and domain A at the core protein position. The resulting final library has a theoretical complexity of 600. (B) Distribution of the implemented modules in accordance to their functional class of a test set of 88 randomly selected clones. (C) Expression titer of the 88 randomly selected clones as determined by ELISA in two independent experiments (Pearson’s Correlation r = 0.8642, R2 = 0.7468, P (two-tailed) <0.0001). Red diamond represents the starting expression level. All clones are correctly assembled and include all functions essential for expression like promoter and signal peptide. (D) Construct design of the Top 4 expression constructs.</p

Expression test library of hPTK7-ECD1-7.

<p>(A) hPTK7-ECD1-7 expression optimization library design. All listed functional modules were included in a single reaction resulting in a theoretical complexity for the final library of 72. (B) Expression test of 20 unique expression clones in deep well expression (DWP) system (y-axis) or in a tube spin expression system (x-axis). Red diamond represents starting expression level of the original construct. Yellow and green diamonds show the expression level of duplicates of the same construct (yet derived from independent E. coli clones) for 2 cases (Pearson’s Correlation r = 0.7380, R2 = 0.5447, P (two-tailed) <0.0001). (C) Construct design of the Top 5 expression constructs in DWP and Tube spin expression.</p

General overview of the MoPET design and implemented functional parts.

<p>(A) Modular structure of the MoPET system consisting of the eight basic module types Promoter, Signal Peptide, N-TAG (N-terminal tag), N-Linker (N-terminal linker), Core Protein, C-Linker (C-terminal linker), C-TAG (C-terminal tag) and the vector. Boxes show the fusion sites separating the modules and indicating the reading frame. (B) Layout of level 0 storage plasmids for the respective module positions and the destination backbones. The level 0 storage plasmids confer resistance to kanamycin and allow blue white selection for cloning purposes. Cloning into this plasmid set can be performed via BpiI, with a final BsaI based assembly in the ampicillin resistant level 1 backbones. (C) Compilation of the functional modules compatible with the MoPET system. DKTH-hFc-His (human IgG1 Fc sequence starting with DKTH), PKSC-hFc-His (human IgG1 Fc sequence starting with PKSC), HSA (human serum albumin), mFc (murine Fc sequence), Avi-tag [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref024" target="_blank">24</a>]. (D) Overview of the functional features of the three basic backbones. P: OriP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref025" target="_blank">25</a>], pA: poly adenylation site, RBG: rabbit beta-globin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref026" target="_blank">26</a>], SV40 Simian virus 40, bGH: bovine growth hormone [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176314#pone.0176314.ref027" target="_blank">27</a>], Neo^R: neomycin resistance, Ap^R: ampicillin resistance, Km^R: kanamycin resistance.</p