Gene design, cloning and protein-expression methods for high-value targets at the Seattle Structural Genomics Center for Infectious Disease

Abstract

Any structural genomics endeavor, particularly ambitious ones such as the NIAID-funded Seattle Structural Genomics Center for Infectious Disease (SSGCID) and Center for Structural Genomics of Infectious Disease (CSGID), face technical challenges at all points of the production pipeline. One salvage strategy employed by SSGCID is combined gene engineering and structure-guided construct design to overcome challenges at the levels of protein expression and protein crystallization. Multiple constructs of each target are cloned in parallel using Polymerase Incomplete Primer Extension cloning and small-scale expressions of these are rapidly analyzed by capillary electrophoresis. Using the methods reported here, which have proven particularly useful for high-value targets, otherwise intractable targets can be resolved.

Figure 1

SSGCID multipronged escalating pipeline. Each Tier can be read from top to bottom, with increasing technology applications read from left to right. Annual goal estimates are tabulated on the right, with Tier-specific success rates calculated along the bottom.

Figure 2

Tier 3 gene design and cloning strategy. (a) Gene Composer design-session window, showing the target amino-acid base construct in green (middle window) and the structure-guided construct-design products in gold (bottom window). (b) Polymerase Incomplete Primer Extension (PIPE) cloning strategy used for this tier of SSGCID pipeline production. Insert PCR products are amplified using primers with homology to the vector termini (shown in red and blue). Vector PCR products are amplified by primers with homology to only the vector termini. (c) Agarose-gel analysis of insert PCR (with target amino-acid numbering) and vector PCR products.

Figure 3

Analysis and quantitation of point-mutant recombinant proteins partially purified on a small scale. (a) Virtual gel of capillary electrophoresis by Caliper LapChip 90. Yields vary by mutant. (b) Mutant specific protein yields obtained, with wild-type protein indicated by a red arrow.

Figure 4

Protein crystal of polymerase PB2 subunit from 2009 pandemic influenza H1N1.

Figure 5

Protein structures of influenza polymerase subunit PB2 from a variety of viral strains obtained using the methods described in this publication. PDB codes (clockwise from top left): 3r2v (T. E. Edwards, A. S. Gardberg & B. Sankaran, unpublished work), 3kc6 (Yamada et al., 2010 ▶), 3l56 (Yamada et al., 2010 ▶) and 3khw (Yamada et al., 2010 ▶).