Improved expression and purification of sigma 1 receptor fused to maltose binding protein by alteration of linker sequence

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Highlights

  • Yield of active MBP-S1R depends on Escherichia coli expression host and domain linker length.

  • E. coli B834-pRARE2 and a 4-Ala linker gave the highest yield of active protein.

  • The fusion protein can be purified in high yield in a mixture of Triton-X100 and DDM.

  • The purified fusion protein has high specific ligand binding activity.

Abstract

Sigma 1 receptor (S1R) is a eukaryotic membrane protein that functions as an inter-organelle signaling modulator and chaperone. Here we report an improved expression of S1R in Escherichia coli as a fusion to maltose binding protein (MBP) and a high-yield purification. Variants with linking amino acid sequences consisting of 0–5 alanine residues between MBP and S1R were created and tested in several E. coli expression strains in order to determine the best combination of construct and host for production of active MBP-S1R. Among the linker variations, the protein containing a 4-Ala linker exhibited superior expression characteristics (MBP-4A-S1R); this construct was most productively paired with E. coli B834-pRARE2 and a chemically defined growth and expression medium. A 3-step purification was developed, including extraction from the E. coli membrane fraction using a mixture of Triton X-100 and n-dodecyl-beta-D-maltopyranoside identified by screening constrainted by retention of binding function, and purification by amylose affinity and gel filtration chromatographies. This procedure yields ∼3.5 mg of purified fusion protein per L of bacterial culture medium. Purified MBP-4A-S1R showed a 175-fold purification from the starting cellular lysate with respect to specific ligand binding activity, and is stable during concentration and freeze–thaw cycling.

Introduction

Sigma 1 receptor (S1R) is a 223 amino acid eukaryotic membrane protein found in the ER membrane of tissues of the endocrine, immune, and nervous systems. S1R interacts with progesterone and testosterone, and a diverse set of compounds including cocaine amphetamines, haloperidol, pentazocine, ditolylguanidine, hallucinogens and others [1], [2], [3]. S1R is implicated in inter-organelle signaling associated with neurological disorders and stroke, regulation of calcium homeostasis in mitochondria, sterol hormone synthesis and the etiology of addiction [4], [5], [6], [7], [8], [9], [10]. Knockout mice, which are otherwise viable, exhibit a variety of changes in psychological responses to stimuli, implicating S1R in pain response, learning, and psychoses [11], [12], [13]. S1R also interacts with other membrane proteins like acid-sensing channel and Nav1.5 voltage-gated Na+ channel, potentially providing modulator or chaperone-like properties [14], [15].

S1R is a member of the ERG2_Sigma1 family (PFAM PF04622), which has a single domain architecture; greater than 100 homologous sequences have been identified in different eukaryotes. The family also includes fungal sterol binding proteins [16]. The primary sequence is highly conserved among different mammals, with >90% identity over 223 residues in human, chimpanzee, mouse, cow, rat, Mongolian gerbil and others [16]. Alternative splicing may produce transcript variants encoding distinct isoforms whose functions are not yet established. Although there are no three-dimensional structures known for this family, a membrane topology model for S1R has been assembled from biochemical and biophysical studies [17], [18]. S1R is thus predicted to be an α-helical membrane protein with two potential transmembrane domains [19].

S1R genes have been cloned from rodents and humans, and the protein has been expressed in Escherichia coli, Saccharomyces cerevisiae and CHO cells [2], [20], [21], [22], [23]. The average yield of purified functional protein, whether from previous recombinant systems or from natural tissues, is low: e.g., 0.2 mg/L from E. coli culture and 0.2 mg from microsomes prepared from guinea pig liver.

Maltose binding protein (MBP) has been fused to membrane proteins in order to promote their expression, purification, and formation of crystals [24], [25], [26], [27], [28], [29], [30]. Moreover, periplasmic export of MBP has been shown to facilitate incorporation of appended membrane protein domains into the bacterial membrane [31], [32], [33]. Correspondingly, previous work showed that an MBP-S1R fusion containing a linker sequence for the Factor Xa protease recognition sequence could be expressed in E. coli BL21(DE3) and a functional form could be obtained, albeit in low yield, 0.6 mg of fusion protein per L of culture medium [2]. Our new results show that the E. coli strain used for expression and the linker region between MBP and S1R play important roles in production of the active form of the receptor. Arising from the improved expression, improved detergent extraction and improved purification, new protocols for MBP-S1R have been developed, and results from these are reported.

Section snippets

Reagents

All reagents were ACS grade unless otherwise specified. All buffers were prepared from deionized and distilled water (18 MΩ) and filtered through a 0.8 μm filter.

Cloning

The guinea pig S1R gene is summarized as Uniprot Q60492. A plasmid encoding guinea pig S1R fused to periplasm-exportable maltose binding protein with a linker including a tobacco etch virus protease recognition site (MBP-TEV-S1R) was used as the template for PCR reactions. This plasmid was derived from MBP-Xa-S1R [1], [2]. The MBP-S1R

Expression construct design

We investigated whether the linker present in MBP-TEV-S1R (Table 2) could be replaced with shorter linkers in order to improve the expression and handling properties. Thus constructs with linkers consisting of 1–5 Ala residues and another construct that contained no additional amino acids between the MBP and S1R domains were produced by PIPE cloning [34], [35]. Because of the length of the TEV protease recognition site, and uncertainty regarding how this sequence would influence the secretion,

Discussion

Here we have presented an improved procedure for expressing and purifying MBP-S1R in a form that is suitable for additional research on biological function, biophysical characterizations, and potentially structure determination. Three key improvements in the methodology for MBP-S1R are summarized here.

Both the yield of fusion protein and the specific ligand binding activity were best in the rare codon supplemented strain E. coli B834-pRARE2. This strain has been successfully used in the Center

Acknowledgments

This work was supported by NIGMS PSI: Biology Network grant U54 GM094584 to B.G.F. The authors thank Dr. Arnold E. Ruoho, Dr. Uyen B. Chu and Dr. Subramaniam Ramachandran (University of Wisconsin-Madison) for providing the MBP-TEV-S1R plasmid used as the starting template in this work, for use of the scintillation counter used in this study, and their insightful discussions on S1R. The authors thank Dr. Emily Beebe (University of Wisconsin-Madison, USA) for generous assistance in protein

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