Plant-based production and characterization of a promising Fc-fusion protein against microgravity-induced bone density loss

Abstract

Microgravity-induced bone loss is a main obstacle for long term space missions as it is difficult to maintain bone mass when loading stimuli is reduced. With a typical bone mineral density loss of 1.5% per month of microgravity exposure, the chances for osteoporosis and fractures may endanger astronauts' health. Parathyroid Hormone or PTH (1-34) is an FDA approved treatment for osteoporosis, and may reverse microgravity-induced bone loss. However, PTH proteins requires refrigeration, daily subcutaneous injection, and have a short shelf-life, limiting its use in a resource-limited environment, like space. In this study, PTH was produced in an Fc-fusion form via transient expression in plants, to improve the circulatory half-life which reduces dosing frequency and to simplify purification if needed. Plant-based expression is well-suited for space medicine application given its low resource consumption and short expression timeline. The PTH-Fc accumulation profile in plant was established with a peak expression on day 5 post infiltration of 373 ± 59 mg/kg leaf fresh weight. Once the PTH-Fc was purified, the amino acid sequence and the binding affinity to its target, PTH 1 receptor (PTH1R), was determined utilizing biolayer interferometry (BLI). The binding affinity between PTH-Fc and PTH1R was 2.30 × 10^-6 M, similar to the affinity between PTH (1-34) and PTH1R (2.31 × 10^-6 M). Its function was also confirmed in a cell-based receptor stimulation assay, where PTH-Fc was able to stimulate the PTH1R producing cyclic adenosine monophosphate (cAMP) with an EC₅₀ of (8.54 ± 0.12) x 10^-9 M, comparable to the EC₅₀ from the PTH (1-34) of 1.49 × 10^-8 M. These results suggest that plant recombinant PTH-Fc exhibits a similar binding affinity and potency in a PTH1R activation assay compared to PTH. Furthermore, it can be produced rapidly at high levels with minimal resources and reagents, making it ideal for production in low resource environments such as space.

Keywords: Nicotiana benthamiana; bone loss; bone regeneration; microgravity; parathyroid hormone; space mission; transient plant expression.

FIGURE 1

The gene construct for transient PTH-Fc expression in Nicotiana benthamiana. RB/LB: right border/left border; LIR: long intergenic region; 35S: Cauliflower mosaic virus 35S promoter; ATADH5’: 5′ UTR of the Arabidopsis thaliana alcohol dehydrogenase gene for translation enhancement; 2S2: secretory signal peptide; PTH-Fc: PTH-Fc coding sequence; HSP: Arabidopsis thaliana HSP 18.2 terminator; SIR: short intergenic region; C1,C2: Rep/RepA coding sequence; NOS promoter/NOS: nopaline synthase promoter/terminator. nptII: gene codes for an aminoglycoside phosphotransferase II conferring resistance to kanamycin for stable transgenic line selection (if desired).

FIGURE 2

PTH-Fc in plant accumulation profile from 1–6 DPI by ELISA (A) and Western blot analysis detecting PTH domain of PTH-Fc in crude plant extracts from 1–6 DPI (B). Error bars represent the standard error of the mean of duplicate measurements.

FIGURE 3

Amino acid sequence of PTH-Fc compared to the control sequence with undetected sequences represented as dashes. The underlined sequence corresponds to 2S2 secretory signal peptide.

FIGURE 4

Western blot analyses detecting PTH (A) and SEKDEL (B) sequences for purified PTH-Fc (lane 1) and a control protein (lane 2, 47.2 kDa) with the SEKDEL C-terminal motif. From left to right: molecular weight ladder, PTH-Fc and a control protein with SEKDEL C-terminal motif.

FIGURE 5

SDS-PAGE analyses of purified PTH-Fc under reducing (R) and non-reducing (NR) conditions.

FIGURE 6

BLI sensorgrams (A–C) obtained from interactions between PTH1R and PTH-Fc, PTH or CMG2-Fc; (D): BLI steady state analysis of receptor binding affinity to PTH-Fc (red) and PTH (blue).

FIGURE 7

Dose-response curve from PTH-Fc or PTH treated CHO-K1 cells expressing PTH1R.