Engineering and identifying supercharged proteins for macromolecule delivery into mammalian cells

Abstract

Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. The potency of functional delivery in some cases can exceed that of other current methods for macromolecule delivery, including the use of cell-penetrating peptides such as Tat and adenoviral delivery vectors. This chapter summarizes methods for engineering supercharged proteins, optimizing cell penetration, identifying naturally occurring supercharged proteins, and using these proteins for macromolecule delivery into mammalian cells.

Figure 1

Electrostatic surface potentials of −30 GFP, stGFP, +36 GFP, and +48 GFP colored from −25 kT/e (red) to +25 kT/e (blue).

Figure 2

UV-illuminated samples of purified GFP variants (“native”), those samples heated 1 min at 100 °C (“boiled”), and those samples subsequently cooled for 2 h at 25 °C (“cooled”).

Figure 3

UV-illuminated samples of His₃₉ GFP (“native”) at different pH values and after those samples were heated 1 min at 100 °C (“boiled”), and subsequently cooled for 2 h at 25 °C (“cooled”).

Figure 4

Properties of supercharged GFP variants. (A) The charge-dependence of supercharged GFP uptake in cultured HeLa cells treated with 200 nM protein for 4 hours at 37 °C. (B) The excitation and emission spectra of blue, cyan, green and yellow fluorescent variants of +36 GFP. The yellow variant is notable, as it has a large stokes shift with a 400 nm absorption, and a 520 nm emission maxima.

Figure 5

His₃₉ GFP penetrates mammalian cells in a pH-dependent manner consistent with the protonation state of its histidine side chains.

Figure 6

Natural supercharged human proteins (NSHPs) deliver active proteins in vitro and in vivo. (A) Percent recombined cells among floxed tdTomato BSR cells incubated with NSHP-Cre fusions as measured by flow cytometry. (B) Adult floxed LacZ mice were injected subretinally with Cre fusion proteins. Recombination results in LacZ activity, which was visualized with X-gal stain (blue) 3 days after injection. (D) Adult floxed LacZ mice injected in the pancreas with Cre fusion proteins exhibit recombination in the exocrine tissues as indicated by LacZ immunostaining (red) 5 days after injection. (E) Adult floxed luciferase mice injected subcutaneously with Cre fusion proteins exhibit recombination in the white adipose tissue as visualized by luminescence 3 days after injection. White adipose tissue was extracted and place to the right of each mouse.

Figure 7

Plot of human proteins expressed from E. coli within the Protein Data Bank. The blue dots represent proteins with positive charge:molecular weight ratios exceeding +0.75/kD.

Figure 8

Optimized supercharged protein fusion architecture for expression, purification and protein delivery.