A simple approach for the preparation of mature human relaxin-3
Graphical abstract
Research highlights
▶ A single-chain precursor of relaxin-3 with a mini C-peptide and a 6×His tag was designed. ▶ The precursor was expressed well in E. coli cells. ▶ After purification, in vitro refolding, and enzymatic digestion, fully active human relaxin-3 was obtained in high yield and at low cost. ▶ Our present work provides a highly efficient approach for the preparation of relaxin-3 as well as its analogues for functional and structural analyses.
Introduction
The insulin superfamily is a group of peptide hormones that share an identical cysteine arrangement pattern but which have diversified biological functions [20]. In human, ten insulin superfamily members have been identified, including insulin, insulin-like growth factor 1 and 2 (IGF-1 and IGF-2), insulin-like peptide 3–6 (INSL3, INSL4, INSL5, and INSL6), and relaxin-1, -2, and -3. Among these, relaxin-3 (also known as INSL7) is the most recently identified. It was identified in 2002 by a search of the human genome based on the cysteine pattern of B-chain of the insulin superfamily [3], [4]. Relaxin-3 has partial activity overlap with relaxin-1 and -2 [3]. It can activate the G-protein coupled receptor RXFP1 (LGR7), the cognate receptor of relaxin-1 and -2 [24]. It was later found that relaxin-3 is also a high affinity ligand for RXFP3 (GPCR135) and RXFP4 (GPCR142) [10], [11]. RXFP3 is thought to be the endogenous receptor of relaxin-3 in brain [11], [12], while RXFP4 does not contribute to relaxin-3 binding in the mouse brain [25]. Relaxin-3 is predominantly expressed in the nucleus incertus of the brain [3], [4], [11], [13], [21]. In the rat model, the relaxin-3 expression level in the nucleus incertus is significantly increased after water-restraint stress [26] or after repeated forced swim stress [1], suggesting that relaxin-3 is involved in the regulation of the stress response. Intracerebroventricular injection of relaxin-3 to rats significantly increases food intake, and chronic relaxin-3 administration increases the body weight [5], [15], [16], [17], suggesting that relaxin-3 is involved in control of food intake. Intracerebroventricular and intraparaventricular administration of human relaxin-3 in adult male rats significantly increased plasma luteinizing hormone, suggesting relaxin-3 plays a role in the stimulation of the hypothalamo-pituitary-gonadal axis [14]. Relaxin-3 knockout mice are healthy with normal body weight, motor coordination, anxiety, spatial memory, and sensorimotor gating. However, female knockout mice display hypoactivity; while male knockout mice display hypersensitivity to stress, suggesting that relaxin-3 signaling contributes to the central control of arousal, exploratory behavior, and stress responses [22], [23].
Relaxin-3 is synthesized in vivo as a precursor that contains a signal peptide, a B-chain, a C-peptide, and an A-chain [3]. After signal peptide removal, correct disulfide pairing, and C-peptide removal, the precursor is converted to the mature two-chain form. Relaxin-3 adopts a relaxin-like fold, but its structure differs crucially from the crystal structure of relaxin-2 in that its B-chain C-terminus folds back, allowing B27Trp to interact with the hydrophobic core and partly blocking the conserved RXXXRXXI motif [18]. Relaxin-3 can be prepared by chemical synthesis of the separate A- and B-chains and subsequent site-directed formation of the three disulfide bonds [2], [27]. It can also be prepared through recombinant expression in mammalian cells [7], [11]. However, the expression level of relaxin-3 in mammalian cells is low, being ∼60 μg/l in one report [7] and ∼1 mg/l in other reports [9], [11]. Mammalian cell culture is also expensive and time consuming. In the present work, we have established a simple approach for the preparation of mature human relaxin-3 through recombinant expression in E. coli cells that grow quickly in cheap medium. After purification, in vitro refolding, and one-step enzymatic cleavage, typically 2–3 mg of fully active human relaxin-3 could be obtained from 1 l of the E. coli culture broth. Our present work thus provides an efficient approach for preparation of relaxin-3 and its analogues.
Section snippets
Materials
The oligonucleotide primers were synthesized at Invitrogen (Shanghai, China). Endoproteinase Asp-N and sequencing grade trypsin were purchased from Sigma–Aldrich (St. Louis, USA). The Agilent reverse-phase columns (analytical column: Zorbax 300SB-C18, 4.6 mm × 250 mm; semi-preparative column: Zorbax 300SB-C18, 9.4 mm × 250 mm) were used in the experiments. The peptide was eluted from the C18 reverse-phase column by an acetonitrile gradient composed of solvent A and solvent B. Solvent A was 0.1% aqueous
Gene construction, expression, and purification of the single-chain relaxin-3 precursor
To prepare human relaxin-3 through recombinant expression in E. coli, we designed a single-chain precursor as shown in Fig. 1A and B. In this precursor, the B- and A-chains of human relaxin-3 were connected by a peptide linker that is rich in hydrophilic residues. This linker was expected to be removed by endoproteinase Asp-N that cleaves the peptide bond at the N-terminal side of Asp residue. A 6×His tag, that would facilitate purification, is fused at the N-terminal of the B-chain. The tag
Acknowledgments
This work was supported by the National Natural Science Foundation of China (30970609) and the National Basic Research Program of China (973 Program, no. 2010CB912604). The studies at the HFI (Melbourne) were supported in part by NHMRC project grants (#508995 and 509048) to JDW and RADB. We thank Linda Chan (FNI) for the amino acid analysis and Tania Ferraro for the activity assays.
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