Prokaryotic Expression And Purification Of Mouse RANKL With An Unnatural Amino Acid Incorporation And Antiserum Preparation

Osteoporosis is a systemic skeletal disease characterized by a progressive loss of bone mass and microarchitectural deterioration of bone tissue, and therefore leads to increased fragility fractures. It is thought to severely affect patient’s quality of life with high incidence and brings a heavy economic burden to the society. Bone homeostasis is maintained by a dynamic balance between osteoblastic bone formation and osteoclastic bone resorption. Excessive activation of osteoclastic bone resorption is a common pathological mechanism of bone loss and fractures.Receptor Activator of Nuclear Factor-κB Ligand(RANKL)plays a key role on differentiation, maturation and function of osteoclast. RANKL binds to the Receptor Activator of Nuclear Factor-κB(RANK), and then activates the downstream signal pathways such as NF-κB, P38, ERK, JNK, etc. and stimulates the differentiation, maturation and function of osteoclasts. The differentiation and maturation of osteoclasts can be inhibited by blocking the binding of RANKL and RANK. It is certified that Denosumab, an anti-RANKL monoclonal antibody, can specifically block the binding of RANKL to its receptors, which inhibits the differentiation, maturation and function of osteoclast effectively. Recent studies show that the genetic incorporation of p-nitrophenylalanine(p NO2Phe), one of unnatural amino acid, into the self-proteins can elicit high titer autoantibody responses to their endogenous proteins.This study was to investigate the prokaryotic expression and purification of mouse RANKL with an unnatural amino acid mutant and preparation of antiserum against m RANKL in mice, and then further to analyze the antibody titer and activity of the antiserum, which established the foundation for the further research on new methods of blocking RANKL-RANK pathway. Methods and Results① Total RNA was extracted from mouse bone marrow, and then the extracellular m RANKL gene was amplified by RT-PCR. p ET28a-p NO2Phe234 m RANKL recombinant expression vector was constructed after mutating the 234 th gene codon(TAT)encoding tyrosine into the amber codon( TAG) which can encode unnatural amino acid p-nitrophenylalanine(p NO2Phe). The recombinant expression vector was verified by sequencing.② The recombinant expression vector(p ET28a-p NO2Phe234 m RANKL) was co-transformed into E.coli BL-21(DE3)with p EVOL plasmid. Protein expression was induced with 0.2 % arabinose and 1 m M IPTG and then purified. SDS-PAGE and Western Blot was used to detect the protein expressed and purified. High performance liquid chromatography(HPLC) in combination with mass spectrometry was conducted to confirm the successful incorporation of an p-nitrophenylalanine( p NO2Phe) into m RANKL.③ Mice were immunized with the purified protein by using the RIMMS(repetitive immunization at multiple sites) protocol. The anti-m RANKL antiserum titer was determined by indirect ELISA, and its specificity was confirmed by Western Blot. The results showed that the mice antiserum against m RANKL was successfully prepared and the titer of antiserum was 1:6400. With high activity, the antiserum not only can be combined with p NO2Phe234 m RANKL, but also can be combined with the wild type m RANKL. ConclusionIn this experiment, we cloned the extracellular m RANKL gene and first constructed p ET28a-p NO2Phe234 m RANKL recombinant expression vector. The protein of m RANKL with a p-nitrophenylalanine mutant was expressed and purified successfully which was confirmed by High performance liquid chromatography(HPLC) in combination with mass spectrometry. Then mice were immunized with the purified protein and anti-m RANKL antiserum with high titer and activity was successfully prepared. This study established the foundation for the further research on new methods of blocking RANKL-RANK pathway and therapying osteoporosis.