# Synthesis of Arapaima gigas Growth Hormone (ag-GH) in HEK 293 Cells: Its Purification and Characterization via In Vivo Bioassay in Dwarf “Little” Mice

## TL;DR

This study shows that a fish growth hormone can be produced in human cells and has similar effects to human growth hormone in mice.

## Contribution

The study demonstrates the successful synthesis and biological activity of ag-GH in a mammalian expression system.

## Key findings

- ag-GH was successfully synthesized and purified from HEK293 cells.
- The purified ag-GH showed a molecular mass close to the theoretical value.
- ag-GH exhibited biological activity comparable to human growth hormone in dwarf mice.

## Abstract

Arapaima gigas growth hormone (ag-GH) cDNA was previously cloned from A. gigas pituitaries. In this work ag-GH has been synthesized using human embryonic kidney 293 cells (HEK293) transiently transfected with the 3.4-TOPO® vector carrying ag-GH cDNA. The 4th day after transfection, the presence of putative ag-GH was detected via SDS-PAGE and Western blotting in comparison with human GH. Ion exchange purification exhibited a clearly symmetric peak, absent in the control medium. The purified fraction, submitted to high-performance size-exclusion chromatography (HPSEC), SDS-PAGE, and Western blotting, contained an immunoreactive molecule, slightly smaller than hGH as expected. MALDI-TOF-MS determined a high-resolution molecular mass of 21,220 Da versus a theoretical value of 21,150. A phylogenetic analysis positioned ag-GH within basal teleost lineages, consistent with earlier analyses of A. gigas gonadotrophic hormones, reinforcing the structural and functional conservation relevant for its biologic activity. An in vivo bioassay based on the body weight increase of dwarf “little” mice demonstrated a biological activity for ag-GH comparable to that of the international reference preparation of rec-hGH. For two species (H. sapiens and A. gigas) separated by an evolutionary period of >100 million years, such a positive biological correlation is remarkable.