A Polypeptide from the Brine Shrimp Artemia Franciscana is Related to Plant Seed Maturation Proteins and Protects Lactate Dehydrogenase During Freezing and Desiccation

Abstract

Several anhydrobiotic organisms accumulate hydrophilic polypeptides, including Late Embryogenesis Abundant (LEA) proteins, before the onset of dehydration. In the brine shrimp A. franciscana, multiple LEA proteins are expressed in the desiccation tolerant life‐history stage, and these proteins localize to different subcellular compartments. The protein AfrLEA6 contains a seed maturation protein (SMP) superfamily domain (pfam04927) and shows homologies to SMP proteins from plants (e.g. Nicotiana sylvestris, XP_009764712.1; e‐value 2e‐04). Even though the expression of LEA proteins is closely related with desiccation tolerance, the precise function and purpose of AfrLEA6 in A. franciscana is unknown. To better understand the role of LEA proteins in desiccation tolerance, large quantities of the protein are needed for functional studies. Therefore, an Escherichia coli expression system was chosen to generate sufficient quantities of AfrLEA6 for in vitro studies. Isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) mediated induction using the vector pTXB1 in BL21 cells (E. coli) yielded approximately 20 mg of AfrLEA6 protein per liter of culture. Intein mediated purification with a chitin‐binding tag allowed for a single affinity chromatography step and avoids the use of additional amino acids that remain on the protein of interest. This system yielded AfrLEA6 at 95% purity after optimization. However, the c‐terminal amino acids penultimate to the beginning of the intein protein played a crucial role in the recovery efficiency using dithiothreitol (DTT) to induce intein cleavage. Maintaining the c‐terminal aspartic acid (Asp) residue found in the native AfrLEA6 protein yielded almost no cleavage when treated with DTT. However, when Asp was mutated to glutamic acid or alanine, cleavage efficiency was greatly improved. To evaluate potential protective properties of AfrLEA6 against freezing, heat‐denaturation, or desiccation induced denaturation, lactate dehydrogenase was chosen as model enzyme. In presence of 25μg ml−1 AfrLEA6 recovery of LDH activity after desiccation was significantly higher compared to control or in presence of lysozyme, BSA or the group 1 LEA protein AfrLEA1.1. In absence of AfrLEA6 and presence of lysozyme or BSA only about 25%‐50% of LDH activity was recovered, while at a AfrLEA6‐to‐LDH mass ratio of 5:1 nearly 70% of enzyme activity was protected. Similarly, 82% recovery of LDH activity was found after liquid nitrogen freezing at a AfrLEA6‐to‐LDH mass ratio of 10:1 while in presence of LEA1.1, BSA, or and lysozyme 64%, 59%, or 40% was recovered. Recovery of LDH activity after heating to 46°C, was also significantly improved in presence of AfrLEA6 compared to the other proteins, but the enzyme lost most of its activity within 30 minutes of temperature challenge under all conditions. Further research to elucidate the mechanism by which AfrLEA6 protects enzymes from denaturation during freezing, drying, and elevated temperature may offer novel avenues for biotechnological applications ranging from cell stabilization to storage of biomedical relevant proteins.Support or Funding InformationThis project was supported by the National Sciences Foundation (IOS‐1659970/IOS‐1457061).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.