In Vitro Recombination of Non-Homologous Genes Can Result in Gene Fusions that Confer a Switching Phenotype to Cells

Abstract

Regulation of protein activity is central to the complexity of life. The ability to regulate protein activity through exogenously added molecules has biotechnological/biomedical applications and offers tools for basic science. Such regulation can be achieved by establishing a means to modulate the specific activity of the protein (i.e. allostery). An alternative strategy for intracellular regulation of protein activity is to control the amount of protein through effects on its production, accumulation, and degradation. We have previously demonstrated that the non-homologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in fusion proteins in which β-lactamase enzyme activity is allosterically regulated by maltose. Here, through use of a two-tiered genetic selection scheme, we demonstrate that such recombination can result in genes that confer maltose-dependent resistance to β-lactam even though they do not encode allosteric enzymes. These ‘phenotypic switch’ genes encode fusion proteins whose accumulation is a result of a specific interaction with maltose. Phenotypic switches represent an important class of proteins for basic science and biotechnological applications in vivo.

Engineering of temperature- and light-switchable Cas9 variants

“…Previous efforts to develop switchable proteins by combining sensor and effector domains (39,40) have yielded both ‘true’ allosteric molecules as well as ‘phenotypic’ constructs. Activity differences of the latter can often be attributed to increased cellular protein accumulation in the ‘on’ condition of the switch (40).…”

“…In the naïve library, light dependence was apparently restricted to two insertion sites, and shifting the paRC9A insertion site by three residues led to high constitutive activity (Supplementary Figure S7), which suggests that the observed behavior requires a particular orientation of Rs LOV relative to Cas9. Initial experiments (Supplementary Figure S13) indicate that differences in protein accumulation (which has been implicated for other phenotypic switches) (39) are not responsible for the observed activity difference. One hypothesis that could explain these data is that the different molecular shape of Rs LOV (5) in the light state causes paRC9 to interact differently with cellular constituents, as has been shown for other proteins (43).…”

The interplay between effector binding and allostery in an engineered protein switch

“…MBP317‐347 accumulates to higher levels in E. coli cells in the presence of maltose as compared to the absence of maltose and does so in a dose‐dependent manner . This phenomenon along with MBP317‐347's allosteric properties contributes to the maltose‐dependent ampicillin resistance phenotype.…”

“…First, MBP317‐347 functions as a monomeric allosteric enzyme, and the allostery is dependent on the maltose‐dependent conformational change in the MBP domain . Second, maltose causes the switch to accumulate to higher levels in the cell . MBP317‐347 has near wildtype affinity for maltose .…”

Protein Switch Engineering by Domain Insertion

“…Many of the MBP–BLA switches function as heterotropic allosteric enzymes, and NMR and crystallographic studies of one switch are consistent with the expectation that the individual domain structures of RG13 are substantially conserved from MBP and BLA (Ke et al, 2012; Wright, Majumdar, Tolman, & Ostermeier, 2010). More recently, we have identified MBP–BLA switch genes that do not encode allosteric proteins but rather encode a protein whose cellular accumulation increases in the presence of the effector, thereby conferring an effector-dependent switching phenotype to cells (Heins, Choi, Sohka, & Ostermeier, 2011; Sohka et al, 2009). Finally, our MBP–BLA switch work has shown how domain fusion can result in emergent properties.…”