Abstract
Biofabrication utilizes biological materials and biological means, or mimics thereof, for assembly. When interfaced with microelectronics, electrobiofabricated assemblies enable exquisite sensing and reporting capabilities. We recently demonstrated that thiolated polyethylene glycol (PEG-SH) could be oxidatively assembled into a thin disulfide crosslinked hydrogel at an electrode surface; with sufficient oxidation, extra sulfenic acid groups are made available for covalent, disulfide coupling to sulfhydryl groups of proteins or peptides. We intentionally introduced a polycysteine tag (5xCys-tag) consisting of five consecutive cysteine residues at the C-terminus of a Streptococcal protein G to enable its covalent coupling to an electroassembled PEG-SH film. We found, however, that its expression and purification from E. coli was difficult, owing to the extra cysteine residues. We developed a redox-based autoinduction methodology that greatly enhanced the yield, especially in the soluble fraction of E. coli extracts. The redox component involved the deletion of oxyRS, a global regulator of the oxidative stress response and the autoinduction component integrated a quorum sensing (QS) switch that keys the secreted QS autoinducer-2 to induction. Interestingly, both methods helped when independently employed and further, when used in combination (i.e., autodinduced oxyRS mutant) the results were best—we found the highest total yield and highest yield in the soluble fraction. We hypothesize that the production host was less prone to severe metabolic perturbations that might reduce yield or drive sequestration of the -tagged protein into inclusion bodies. We expect this methodology will be useful for the expression of many such Cys-tagged proteins, ultimately enabling a diverse array of functionalized devices.
Highlights
Affinity tags incorporated into the primary sequences of recombinant proteins were initially developed as a means to facilitate their purification and/or detection (Terpe, 2003; Kimple et al, 2013)
We designed a polycysteine tag inserted at the C-terminus of the target protein and retained the N-terminus His-tag found in many commercial expression vectors for purification purposes (Figure 2)
Note that surfaces without the protein G-5xCys layer exposed identically to E. coli did not retain cells. While this is largely a result of the specificity of the anti-E. coli antibody, it reflects the minimal level of non-specific binding attributed to the polyethylene glycol (PEG) hydrogel, consistent with many reports regarding the properties of PEGylated surfaces
Summary
Affinity tags incorporated into the primary sequences of recombinant proteins were initially developed as a means to facilitate their purification and/or detection (Terpe, 2003; Kimple et al, 2013). Because protein engineering is fairly routine, the incorporation of “designer” tags has emerged enabling a variety of new functions, among those including protein attachment onto both. Methodologies that allow protein attachment to various substrates have created new possibilities to construct devices with diverse functions introduced by the assembled proteins. These “designer” proteins, can present challenges in expression and purification, owing to the added tags (Kim et al, 2012; Lilie et al, 2013); yet their value is worth the challenge
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