Purification of enzymatically active APOBEC proteins from an insect cell expression system

Abstract

The APOBEC cytidine/deoxycytidine deaminase family of enzymes has 11 members in humans. These enzymes carry out essential developmental, metabolic, and immunological functions through the deamination of cytosine to form uracil in RNA or single-stranded DNA. The known physiological functions relate to lipid absorption (APOBEC1), immunoglobulin gene diversification (AID), virus restriction (APOBEC3A-H, excluding E), and muscle differentiation (APOBEC2). The ability to characterize in vitro how APOBEC enzymes interact with and catalyze cytidine/deoxycytidine deamination of their substrate has provided key insights and understanding of their physiological functions. Having the most highly active and soluble enzyme to carry out in vitro experiments is essential. For APOBEC enzymes this requires purification from a mammalian or insect cell system. Since mammalian cell expression is lower than robustly engineered recombinant systems such as the Spodoptera frugiperda 9 (Sf9) and baculovirus systems, we have developed recombinant baculovirus expression and purification methods for APOBEC enzymes from Sf9 cells. The yield for all family members is suitable for biochemical assays, with some enzymes yielding milligram amounts (suitable for structural studies). Here we describe the expression and purification of APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3F, APOBEC3G, APOBEC3H (Haplotypes II, V, VII), and APOBEC1 using existing molecular biology reagents. We also describe how to clone a novel gene into the system for expression and purification. Due to different expression levels and solubility, three purification methods are detailed that enable high, medium, and low expressing APOBECs to be purified.

Keywords: AID/APOBEC enzymes; Affinity purification; Baculovirus; Cytosine deaminase; Sf9 cells.