Abstract

Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.

Highlights

  • The immune response to various intracellular pathogens and tumors includes cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells which recognize and directly kill infected or malignant cells

  • We suggest performing the transfection in 10 plates at a time to ensure that the timing between DNA-lipid complexing and addition to plates does not extend over 15 minutes

  • The purification in high yields of cytotoxic granular proteins in a reliable and consistent manner has been a significant barrier in the field of immunology

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Summary

Introduction

The immune response to various intracellular pathogens and tumors includes cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells which recognize and directly kill infected or malignant cells. Due to its proinflammatory potential, there have been recent reports of its involvement during bacterial sepsis [16, 18, 19] To further study these effects, it is imperative that researchers are careful to avoid potential endotoxin contamination in the final purified products: the use of a bacterial expression system will directly contaminate purified proteins, while any other system will contaminate the product if the researcher is not careful throughout the process. In contrast to human GzmA, Granzyme B (GzmB) induces apoptosis of the target cells either by direct or indirect activation of caspase 3 and 7 [20, 21]. Activated caspases trigger the release of an active DNase (CAD), responsible for DNA fragmentation and nuclear changes during apoptosis [24]

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