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Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli.

ABSTRACT: Receptor activator of nuclear factor (NF)-?B ligand (RANKL), a master cytokine that drives osteoclast differentiation, activation and survival, exists in both transmembrane and extracellular forms. To date, studies on physiological role of RANKL have been mainly carried out with extracellular RANKL probably due to difficulties in achieving high level expression of functional transmembrane RANKL (mRANKL). In the present study, we took advantage of codon optimization and response surface methodology to optimize the soluble expression of mRANKL in E. coli. We optimized the codon usage of mRANKL sequence to a preferred set of codons for E. coli changing its codon adaptation index from 0.64 to 0.76, tending to increase its expression level in E. coli. Further, we utilized central composite design to predict the optimum combination of variables (cell density before induction, lactose concentration, post-induction temperature and post-induction time) for the expression of mRANKL. Finally, we investigated the effects of various experimental parameters using response surface methodology. The best combination of response variables was 0.6 OD600, 7.5 mM lactose, 26°C post-induction temperature and 5 h post-induction time that produced 52.4 mg/L of fusion mRANKL. Prior to functional analysis of the protein, we purified mRANKL to homogeneity and confirmed the existence of trimeric form of mRANKL by native gel electrophoresis and gel filtration chromatography. Further, the biological activity of mRANKL to induce osteoclast formation on RAW264.7 cells was confirmed by tartrate resistant acid phosphatase assay and quantitative real-time polymerase chain reaction assays. Importantly, a new finding from this study was that the biological activity of mRANKL is higher than its extracellular counterpart. To the best of our knowledge, this is the first time to report heterologous expression of mRANKL in soluble form and to perform a comparative study of functional properties of both forms of RANKL.

SUBMITTER: Maharjan S

PROVIDER: S-EPMC4014495 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli.

Maharjan Sushila S Singh Bijay B Bok Jin-Duck JD Kim Jeong-In JI Jiang Tao T Cho Chong-Su CS Kang Sang-Kee SK Choi Yun-Jaie YJ

PloS one 20140508 5

Receptor activator of nuclear factor (NF)-κB ligand (RANKL), a master cytokine that drives osteoclast differentiation, activation and survival, exists in both transmembrane and extracellular forms. To date, studies on physiological role of RANKL have been mainly carried out with extracellular RANKL probably due to difficulties in achieving high level expression of functional transmembrane RANKL (mRANKL). In the present study, we took advantage of codon optimization and response surface methodolo ...[more]

PMID: 24809485

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Project description:BackgroundLeukemia inhibitory factor (LIF) is a multifunctional cytokine which plays multiple roles in different biological processes such as implantation, bone remodeling, and hematopoiesis. The buESCs are difficult to culture due to lack of proper understanding of the culture conditions. LIF is one of the important factors which maintain the pluripotency in embryonic stem cells and commercial LIF from murine and human origin is used in the establishment of buffalo embryonic stem cells (buESCs). The LIF from a foreign origin is not able to maintain pluripotency and proliferation in buESCs for a long term which is contributed by difference in the binding sites on LIF; therefore, culture medium supplemented with buffalo-specific LIF may enhance the efficiency of buESCs by improving the environment of culture conditions. The high cost of LIF is another major drawback which restricts buESCs research, thus limits the scope of buffalo stem cell use. Various methods have been developed to produce human and murine LIF in prokaryotic system. However, Buffalo leukemia inhibitory factor (BuLIF) has not been yet produced in prokaryotic system. Here, we describe a simple strategy for the expression and purification of biologically active BuLIF in Escherichia coli (E. coli).ResultsThe BuLIF cDNA from buffalo (Bubalus bubalis) was cloned into pET22b(+) and expressed in E. coli Lemo-21(DE3). The expression of BuLIF was directed into periplasmic space of E. coli which resulted in the formation of soluble recombinant protein. One step immobilized metal affinity chromatography (IMAC chromatography) was performed for purification of BuLIF with ≥ 95% of homogeneity. The recombinant protein was confirmed by western blot and identified by mass spectroscopy. The biological activity of recombinant BuLIF was determined on murine myeloid leukemic cells (M1 cells) by MTT proliferation assay. The addition of BuLIF increased the reduction of MTT by stimulated M1 cells in a dose-dependent manner. The BuLIF induced the formation of macrophage like structures from M1 cells where they engulfed fluorescent latex beads. The recombinant BuLIF successfully maintained pluripotency in buffalo embryonic stem cells (buESCs) and were positive for stem cells markers such as Oct-4, Sox-2, Nanog, and alkaline phosphatase activity.ConclusionsThe present study demonstrated a simple method for the production of bioactive BuLIF in E. coli through single step purification. BuLIF effectively maintained buffalo embryonic stem cells pluripotency. Thus, this purified BuLIF can be used in stem cell study, biomedical, and agricultural research.

Project description:Octamer-binding transcription factor 4 (OCT4) is vital for early embryonic development and is a master regulator of pluripotency in embryonic stem cells. Notably, OCT4 is a key reprogramming factor to derive induced pluripotent stem cells, which have tremendous prospects in regenerative medicine. In the current study, we report heterologous expression and purification of human OCT4 in E. coli to produce pure recombinant protein under native conditions. To achieve this, the 1083 bp coding sequence of the human OCT4 gene was codon-optimized for heterologous expression in E. coli. The codon-optimized sequence was fused with fusion tags, namely a cell-penetrating peptide sequence for intracellular delivery, a nuclear localization sequence for intranuclear delivery, and a His-tag for affinity purification. Subsequently, the codon-optimized sequence and the fusion tags were cloned in the protein expression vector, pET28a(+), and transformed into E. coli strain BL21(DE3) for expression. The recombinant OCT4 protein was purified from the soluble fraction under native conditions using immobilized metal ion affinity chromatography in a facile manner, and its identity was confirmed by Western blotting and mass spectrometry. Furthermore, the secondary structure of the recombinant protein was analyzed using far ultraviolet circular dichroism spectroscopy, which confirmed that the purified fusion protein maintained a secondary structure conformation, and it predominantly composed of α-helices. Next, the recombinant OCT4 protein was applied to human cells, and was found that it was able to enter the cells and translocate to the nucleus. Furthermore, the biological activity of the transduced OCT4 protein was also demonstrated on human cells. This recombinant tool can substitute for genetic and viral forms of OCT4 to enable the derivation of integration-free pluripotent cells. It can also be used to elucidate its biological role in various cellular processes and diseases and for structural and biochemical studies.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-021-02758-z.

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Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli.

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Exploring codon optimization and response surface methodology to express biologically active transmembrane RANKL in E. coli.

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