# Plant-Derived Hydrolysates Are a Suitable Replacement for Tryptone N1 in Recombinant Protein Expression Using Human Embryonic Kidney (HEK293-6E) Cells

## TL;DR

This study shows that plant-based hydrolysates can replace animal-derived Tryptone N1 in HEK293 cells for producing recombinant proteins, offering a sustainable alternative.

## Contribution

The novelty is demonstrating that plant-derived hydrolysates from pea and broad bean are viable and sustainable alternatives to animal-derived Tryptone N1 for recombinant protein expression.

## Key findings

- Plant-derived hydrolysates from pea and broad bean showed similar cell viability as Tryptone N1.
- All three hydrolysates (pea, broad bean, and TN1) produced comparable recombinant protein expression levels.
- Plant-based hydrolysates outperformed the no-hydrolysate control in both viability and protein expression.

## Abstract

Human embryonic kidney (HEK293) cells are a widespread choice for recombinant protein expression. To optimise yields, the hydrolysate Tryptone N1 (TN1) is commonly added post-transfection. TN1 is obtained by controlled enzymatic digestion of casein. As an animal by-product, TN1 faces stricter regulations during cross-country shipments than plant-based products. This raises the question of whether plant-derived peptides are a suitable alternative to TN1. Using polyethyleneimine (PEI) as a cationic polymer, we transfected HEK293-6E cells grown in suspension in serum-free medium and divided the transfectants into four groups (each in triplicate). Two plant-based hydrolysates each derived from pea and broad bean were compared with TN1 and a no-hydrolysate control group. We monitored the cultures for total cell numbers and viability at days 1, 4, and 5 post-transfection. Both plant-based hydrolysates and TN1 showed similar live cell percentages, in contrast to the no-hydrolysate control, which showed lower viability. Five days post-transfection, the expressed His-tagged protein, a tegumental antigen from the eukaryotic parasite Echinococcus granulosus, was retrieved from the serum-free culture supernatant, and the expressed recombinant protein was quantified. The linear ranges for the protein load on the stain-free blot and for the use of the fluorescent anti-His-Tag Alexa488 antibody were determined. Using these parameters, stain-free Western blotting and total protein normalization were performed. The plant-derived pea and broad bean hydrolysates reproducibly resulted in similar expression levels as animal-derived TN1; all three hydrolysates were better than no hydrolysate. We conclude that plant-derived hydrolysates are a suitable, more sustainable replacement for TN1.