Genetic Fusion of Transacting Activator of Transcription Peptide to Cyclized Green Fluorescence Protein Improves Stability, Intracellular Delivery, and Tumor Retention

Abstract

Therapeutic proteins such as enzymes, hormones, and cytokines suffer from poor stability, inefficient cellular penetration, and rapid clearance from circulation. Conjugation with polymers (such as poly(ethylene glycol)) and fusion with long-acting proteins (such as albumin and Fc fragments) have been utilized to partially address the delivery issues, but these strategies require the introduction of new macromolecular substances, resulting in potential immunogenicity and toxicity. Herein, we report an easy strategy to increase the intracellular delivery efficiency and stability of proteins by combining of sortase-mediated protein cyclization and cell-penetrating peptide (CPP)-mediated intracellular delivery. We, for the first time, genetically constructed a green fluorescence protein (GFP) fused with a CPP, a transacting activator of transcription (TAT) peptide, at its C-terminus for intracellular internalization, and two sortase recognition sequences, pentaglycine and LPETG, at its N- and C-termini for cyclization. Notably, the cyclized GFP-TAT (cGFP-TAT) not only highly retained the photophysical properties of the protein but also significantly improved the in vitro stability compared with the native linear GFP (lGFP) and linear TAT peptide-fused GFP (lGFP-TAT).Moreover, cGFP-TAT showed better cellular internalization ability compared with lGFP. In C26 tumor-inoculated mice, cGFP-TAT exhibited enhanced in vivo tumor retention, with increases of 7.79- and 6.52-fold relative to lGFP and lGFP-TAT in tumor retention 3 h after intratumor administration. This proof-of-concept study has provided an easy strategy to increase the in vitro stability, intracellular delivery efficiency, and in vivo tumor retention of GFP, which would be applicable to numerous therapeutic proteins and peptides for clinical practice.

Figure 1

Synthesis and characterization of cGFP-TAT, lGFP-TAT, and lGFP. (a) SDS-PAGE analysis. Lane 1: lGFP; lane 2: lGFP-TAT; lane 3: cGFP-TAT. (b) MALDI-TOF spectra of proteins. (c) Fluorescence spectra of proteins from 480 to 540 nm, excited at 460 nm. (d) UV–vis absorption spectra of proteins from 230 to 600 nm.

Scheme 1. Schematic Illustration of the Biosynthesis Process of cGFP-TAT. First, lGFP Was Selectively Fused with a CPP, the TAT Peptide, at Its C-Terminus Using Protein Engineering Technology to Form lGFP-TAT. Second, lGFP-TAT Was Cyclized to Produce cGFP-TAT for Subsequent Sortase A-Catalyzed Ligation

Figure 2

In vitro thermal stability of cGFP-TAT, lGFP-TAT, and lGFP. (a) Fluorescence retention of proteins heated at 80 °C for the indicated times. P < 0.005 for cGFP-TAT vs lGFP and lGFP-TAT. (b) Fluorescence retention percentage of proteins heated at 80 °C for 15 min. P < 0.001 for cGFP-TAT vs lGFP and lGFP-TAT. (c) Fluorescence recovery of proteins following thermal denaturation at 90 °C for 5 min. P < 0.0001 for cGFP-TAT vs lGFP and lGFP-TAT. (d) Fluorescence recovery percentage of proteins 30 min after thermal denaturation. P < 0.0001 for cGFP-TAT vs lGFP and lGFP-TAT. (e) DSC curve of proteins. (f) T_m value of proteins. P < 0.01 for cGFP-TAT vs lGFP and lGFP-TAT. Data are shown as the mean ± standard deviation (n = 3).

Figure 3

In vitro proteolytic and chemical stability of cGFP-TAT, lGFP-TAT, and lGFP. (a) Fluorescence retention of proteins against papain as a function of time. (b) Fluorescence retention percentage of proteins after incubating with papain for 72 h. (c) Fluorescence retention of proteins after incubating in a 6 M GdnHCl solution as a function of time. (d) Fluorescence retention percentage of proteins after incubating in a GdnHCl solution for 45 min. Data are shown as the mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for cGFP-TAT and lGFP-TAT vs lGFP.

Figure 4

Intracellular uptake of cGFP-TAT, lGFP-TAT, and lGFP. (a) Transduction of proteins to C26 cells. C26 cells incubated without GFP were used as the control. The cell nucleus was stained with Hoechst 33342 in blue; the plasma membrane was stained with wheat germ agglutinin in red; and GFP was shown in green with a final incubation concentration of 30 μM at 37 °C in the fresh RPMI-1640 medium. (b) Quantification of the mean fluorescence units in the cell, plasma membrane, and nucleus after taken up by C26 for 1 h (up) and 4 h (below). Data are shown as the mean ± standard deviation (n = 9). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 for lGFP-TAT and cGFP-TAT vs lGFP in the cell, plasma membrane, and nucleus, respectively.

Figure 5

In vivo tumor retention of cGFP-TAT, lGFP-TAT, and lGFP. (a) In vivo fluorescence imaging of proteins in tumor-bearing mice after intratumor injection from 1 min to 3 h. From top to bottom: lGFP, lGFP-TAT, and cGFP-TAT. (b) Fluorescence of proteins in tumors as a function of time. Data are shown as the mean ± standard deviation (n = 4). **P < 0.01 and ****P < 0.001 for cGFP-TAT vs lGFP and lGFP-TAT.