Gene Composer in a structural genomics environment

Abstract

The structural genomics effort at the Seattle Structural Genomics Center for Infectious Disease (SSGCID) requires the manipulation of large numbers of amino-acid sequences and the underlying DNA sequences which are to be cloned into expression vectors. To improve efficiency in high-throughput protein structure determination, a database software package, Gene Composer, has been developed which facilitates the information-rich design of protein constructs and their underlying gene sequences. With its modular workflow design and numerous graphical user interfaces, Gene Composer enables researchers to perform all common bioinformatics steps used in modern structure-guided protein engineering and synthetic gene engineering. An example of the structure determination of H1N1 RNA-dependent RNA polymerase PB2 subunit is given.

Figure 1

The SSGCID structure-determination pipeline. Tiers are represented as columns and described in the text at the bottom of the figure. Rows show the processes through which targets pass in each Tier. The numbers indicate how many targets are expected to pass through each step in the pipeline.

Figure 2

Alignment of influenza A RNA-dependent RNA polymerase PB2 subunit sequences from various influenza A strains with PDB entry 2vy6 (Guilligay et al., 2008; Tarendeau et al., 2008 ▶).

Figure 3

Construct design of InvaB.07055.c.

Figure 4

DNA-sequence modifications available to the user during the gene-design process (Lorimer et al., 2009 ▶).

Figure 5

Construct Design Viewer. In the lower panel the user can make N- or C-terminal deletions, amino-acid insertions, deletions or surface mutations.

Figure 6

Virtual cloning in Gene Composer. Once contructs have been created as shown in Fig. 5 ▶ they can be virtually cloned. The vector is chosen from a pull-down menu and the appropriate adapters added to facilitate the cloning strategy. In this example a stop codon has been added to the end of all the constructs and BamHI and HindIII adapters have been added.

Figure 7

Structure model of influenza A RNA-dependent RNA polymerase subunit PB2 (PDB entry 3kc6; Yamada et al., 2010 ▶). The expressed construct was designed using Gene Composer as described in this paper. The expression and purification methods are as described in Raymond et al. (2011 ▶). The figure was downloaded from the PDB.