Fast and easy protocol for the purification of recombinant S‐layer protein for synthetic biology applications

Abstract

A goal of synthetic biology is to make biological systems easier to engineer. One of the aims is to design – with nanometer-scale precision – biomaterials with well-defined properties. The surface layer protein SbpA forms two-dimensional (2D) arrays naturally on the cell surface of Lysinibacillus sphaericus but also as purified protein in solution upon addition of divalent cations. Its high propensity to form crystalline arrays, the simple way by which its crystallization can be controlled by divalent cations and the possibility to genetically alter the protein make SbpA an attractive molecule for synthetic biology. To be a useful tool, however, it is important that a simple protocol can be used to produce recombinant wild-type as well as modified SbpA in large quantities and in a biologically active form. The present study addresses this requirement by introducing a mild and non-denaturing purification protocol to produce milligram quantities of recombinant, active SbpA.

Controlled and Stable Patterning of Diverse Inorganic Nanocrystals on Crystalline Two-Dimensional Protein Arrays

“…Recombinant His 6 -tagged protein was isolated as previously described by gravity-flow immobilized metal-affinity chromatography. 52 Purified protein was then dialyzed against 20 mM Tris (pH 8.0) to remove imidazole. All purification steps for the isolation of the SbsB Cys cluster also included 10 mM TCEP, to avoid the formation of disulfide linkages between monomers.…”

Programmable assembly of 2D crystalline protein arrays into covalently stacked 3D bionanomaterials

“…When the cell density reached 0.4–0.6 (2–4 hr), protein expression was induced using 1 mM isopropyl β‐ d ‐1‐thiogalactopyranoside. Cells were further grown overnight at 18℃ to avoid the formation of inclusion bodies as previously described for SbpA (Norville, Kelly, Knight, Belcher, & Walz, 2011). Cultures were centrifuged at 6,000 g for 20 min at 4℃ to pellet the cells.…”

“…Recombinant His‐tagged SbsB was isolated from the cell pellet by lysis followed by gravity‐flow immobilized metal‐affinity chromatography as previously described (Norville et al, 2011). The purified SbsB was then dialyzed into 20 mM Tris pH 8.0 to remove imidazole.…”

Meeting report: The Cambridge BioDesign TechEvent – Synthetic Biology, a new “Age of Wonder”?

“…Synthetic biology applications on Earth and beyond: generation of novel, clinically or industrially interesting materials (e.g. rebuilding bone for clinical purposes) using the tools of synthetic biology [5] was also discussed at the meeting. Dr. Michelle Oyen (University of Cambridge, UK) presented her research, which utilizes simple robots to automate integration of organic components into inorganic crystals, to form novel biocomposites that resemble natural materials with high stiffness‐density ratios, such as bone and eggshell.…”