Expression in Escherichia coli, purification and characterization of LRSAM1, a LRR and RING domain E3 ubiquitin ligase

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Highlights

  • LRSAM1 was expressed at high level in Escherichia.coli as inclusion bodies.

  • LRSAM1 was purified from inclusion bodies through denaturation, renaturation and ammonium sulfate precipitation steps.

  • The E3 activity of LRSAM1 was pH-dependent in cooperation with UbcH5-type E2-enzymes.

  • LRSAM1-driven ubiquitination favored K6-, K27-, K29- and K48-linkages.

Abstract

LRSAM1 is a typical RING-finger E3 ubiquitin ligase that plays an important role in many processes. The expression and purification of LRSAM1 from Escherichia coli had not yet been reported. Here, strategies to clone, express and purify recombinant LRSAM1 in E. coli cells were developed. LRSAM1 was expressed with high yield as inclusion bodies and successfully recovered in soluble form by subsequent denaturation and renaturation steps. Refolded LRSAM1 was directly purified through two steps of ammonium sulfate precipitation, resulting in a purity of up to 95% and a yield of about 6 mg/L bacterial culture. Purified recombinant LRSAM1 exhibited a pH-dependent E3 ligase activity. Its ligase activity was RING-finger domain-dependent, and its ubiquitination favors K6-, K27-, K29- and K48-linkages in cooperation with UbcH5-type E2 enzymes.

Introduction

E3 ubiquitin ligases are classified into two major families: (1) the HECT domain family and (2) the RING-finger domain (including RING-like E3 ligases, e.g., U-Box and PHD domains) family [1]. There are approximately 600 RING-finger ligases in the human genome. They play vital roles in cellular signaling processes and are involved in controlling human disease [2]. Human leucine-rich repeat and sterile alpha motif containing 1 (LRSAM1) is classified as a typical RING-finger E3 ubiquitin ligase. The full-length cDNA of LRSAM1 predicts six N-terminal leucine-rich repeat (LRR) domains, two coiled-coil (CC) domains, one sterile-alpha motif (SAM), two Pro-(Ser/Thr)-Ala-Pro (PTAP) tetrapeptide motifs and a C-terminal C3HC4-type RING-finger domain [3], [4].

The E3 ligase activity of LRSAM1 plays an important role in many processes. TSG101, its only known substrate, can be mono-ubiquitinated at many sites by LRSAM1 to regulate vesicular trafficking processes [4], [5], [6]. A report has shown that LRSAM1 also polyubiquitinates the C-terminal lysine residues of TSG101, resulting in its proteasomal degradation and thus controlling the levels of TSG101 [4]. A number of studies have shown that LRSAM1 plays a vital role in the proper adhesion of neuronal cells in culture, receptor protein sorting and membrane vesicle fusion [7]. Mutations in the gene encoding LRSAM1 have been confirmed to cause axonal Charcot-Marie-Tooth (CMT) [5], [6], [7], [8]. The ubiquitin system plays a key role in mediating autophagy degradation of ubiquitin-coated bacteria [9]. Most recently, LRSAM1 was identified as an E3 ligase responsible for the ubiquitin-dependent autophagy of intracellular pathogenic bacteria [10], [11]. The LRSAM1 LRR domain is required for the recognition of intracellular bacteria, and its E3 ligase activity is necessary for defense against invasive pathogens [10], [11], [12], [13]. These findings have inspired us to investigate how LRSAM1 targets its unique substrates because more experiments are required to reveal its molecular mechanism. As such, the production of large amounts of high quality protein is required for both biochemical and biophysical experiments.

In this paper, we describe the purification of recombinant LRSAM1 with excellent activity using a bacterial expression system. The RING finger E3 ligase-driven ubiquitination favored K6-, K27-, K29- and K48-linkages in cooperation with UbcH5-type E2 enzymes.

Section snippets

Plasmids, antibodies and reagents

LRSAM1 cDNA was amplified from a HeLa cDNA library and cloned separately into the pGEX-6p-2, pSUMO, pET-28a, pET-22b and pET-21a vectors. The primers used for construction of plasmids were listed in Table S2. The point mutation constructs of LRSAM1 and ubiquitin-K-only (only one lysine residue remaining and others were substituted by arginine residues) were carried out using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). The original ubiquitin-K0 (all seven lysine residues were

Expression of recombinant LRSAM1

The cDNA of LRSAM1 was cloned into various vectors; the types and sizes of the fusion tags that were used for affinity purification and to enhance solubility are listed in Table S1. The different constructs were expressed in the standard E. coli strain BL21 (DE3). High level expression was obtained for all six constructs. However, the major fractions of expressed proteins were in inclusion bodies, even when the expression temperature was lowered to 16 °C (Fig. 1). There were no visible soluble

Discussion and conclusion

LRSAM1 is a typical RING-finger E3 ligase, and its function is dependent on its E3 ligase activity. Akin to the role of parkin in mitophagy [10], [20], recent reports have shown that LRSAM1 plays an important role in recognizing and targeting intracellular bacteria for autophagic degradation [2], [10], [11], [20].

To study protein function in vitro, the key is to robustly produce high quality protein. Recombinant LRSAM1 had previously been purified from FreeStyle 293-F cells [10]. However, the

Acknowledgements

We thank Dr. Feng Shao from National Institute of Biological Sciences for kindly gift of the pET-28a-Hub-K0 plasmid that was used in this study. We would like to thank Shengyin Liu and Longxiang Xie for helpful discussions. This work was supported by the Natural Science Foundation of China (30970630 and 31270831), the Outstanding Youth Science Foundation of Chongqing (cstc2011jjjq10003), the Program for New Century Excellent Talents in University (NCET-08-0912), the Fundamental Research Funds

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These authors contributed equally to this work.

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