Production and characterization of homologous protoporphyrinogen IX oxidase (PPO) proteins: Evidence that small N-terminal amino acid changes do not impact protein function

Abstract

Transgenic soybean, cotton, and maize tolerant to protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides have been developed by introduction of a bacterial-derived PPO targeted into the chloroplast. PPO is a membrane-associated protein with an intrinsic tendency for aggregation, making expression, purification, and formulation at high concentrations difficult. In this study, transgenic PPO expressed in three crops was demonstrated to exhibit up to a 13 amino acid sequence difference in the N-terminus due to differential processing of the chloroplast transit peptide (CTP). Five PPO protein variants were produced in and purified from E. coli, each displaying equivalent immunoreactivity and functional activity, with values ranging from 193 to 266 nmol min-1 mg-1. Inclusion of an N-terminal 6xHis-tag or differential processing of the CTP peptide does not impact PPO functional activity. Additionally, structural modeling by Alphafold, ESMfold, and Openfold indicates that these short N-terminal extensions are disordered and predicted to not interfere with the mature PPO structure. These results support the view that safety studies on PPO from various crops can be performed from a single representative variant. Herein, we report a novel and robust method for large-scale production of PPO, enabling rapid production of more than 200 g of highly active PPO protein at 99% purity and low endotoxin contamination. We also present a formulation that allows for concentration of active PPO to > 75 mg/mL in a buffer suitable for mammalian toxicity studies.

Fig 1

A: Schematic of various PPO constructs produced and tested. B: Western blot analysis of PPO protein extracted from plant sources. Lanes 1, 4 & 7: MagicMark XP Western Protein Standards (Thermo Fisher Scientific); Lanes 2 & 3: wild-type and PPO-transgenic maize seeds, respectively; Lanes 5 & 6: wild-type and PPO-transgenic cotton seeds, respectively; Lanes 8 & 9: wild-type and PPO-transgenic soybean leaf tissues, respectively. Arrows on blot indicate specific PPO proteins (S1 Raw images). C: N-terminal sequence of plant produced PPO.

Fig 2. SDS PAGE analysis of PPO protein produced from E. coli.

PPO proteins were resolved by SDS-PAGE. Each PPO variant was loaded in its respective lane with one μg of protein. Lanes 1 & 7: molecular weight markers (Precision Plus Protein Dual color, Bio-Rad), which were used to determine the apparent molecular weight of PPO variants; lane 2: Tag-free PPO; lane 3: PPO; lane 4: cPPO; lane 5: sPPO; lane 6: mPPO (S1 Raw images).

Fig 3. Western blot analysis of PPO protein produced from E. coli.

PPO proteins were resolved on a pre-cast Tris-Glycine 4–20% (w/v) polyacrylamide gradient mini-gel by Tris-Glycine-SDS running buffer (Invitrogen, Carlsbad, CA) and electro-transferred onto a nitrocellulose membrane. The blot was probed with an anti-PPO specific mAb and developed using an enhanced chemiluminescence system. Either 0.75 or 1.5 ng of each PPO variant was loaded in each lane containing PPO protein. Lanes 1 and 12: MagicMark XP Western Protein Standards (Thermo Fisher Scientific); lanes 2 and 3: Tag-free PPO; lanes 4 and 5: PPO; lanes 6 and 7: cPPO; lanes 8 and 9: sPPO; lanes 10 and 11: mPPO (S1 Raw images).

Fig 4. Structure alignment of PPO variants.

The structures modeled using Alphafold are presented as representatives. A: Individual PPO variant structure; B: Structure alignment of PPO variants with the tag-free PPO. All align with RMSD of <0.3 Å.