Expression Of Recombinant FL In E.coli And Identification Of Its Bioactivity

FL (Flt3 ligand), a type I transmembrane protein containing two potential sites for N-linked glycosylation, is ubiquitously expressed in human tissues. Although multiple isoforms of FL have been identified, the predominant isoform in human is the transmembrane protein, which can be cleaved proteolytically to yield a soluble, biologically active form. The first 134 residues of amino acids are necessary for FL biological function. FL engaging with Flt3 receptor affects the growth and differentiation of progenitor and stem cells by promoting progenitor cells to enter cell cycles, consequently enhancing the haematopoietic effects. In addition, FL synergies with a wide range of colony stimulating factors (CSFs), such as SCF, G-CSF, GM-CSF and IL-3, to promote the growth and colony formation of both primitive and committed progenitor cells. FL has profound effects on lymphopoiesis and a less potent effect on myelopoiesis. FL is instrumental in long-term reconstitution of haematopoietic stem cells and thus maybe of great value in haematopoietic mobilization and recovery of normal haematopoietic roles after radiation injury and treatments of malignant tumors. Of particular interest and significance is its capacity to upregulate markedly production of DC populations in vivo. FL induces immune tolerance or enhances immune response by promoting production of different populations of DC. The properties of FL make it a promising agent for use in biological/immunotherapy approaches to the treatment of cancer and infectious disease. It is also of value both as an investigative tool and a potential therapeutic agent in tansplantation.Although FL is of therapeutic value as an agent regulating haematopoietic effects, its application is greatly limited due to low-level expression in E. Coli. and poor biological function of products according to present reports. Additionally, expression in other systems is also low, thus producing great difficulty for purification. In our study, toenhance expression level in E.Coll, an artificial FL gene was adopted as template to amplify the target fragment. High expression and high purity of target protein were achieved after denaturation, refolding and one-step purification. Biological function of recombinant protein rhFL134 was characterized by classical colony formation and CD34+ cell proliferation assays. The study provides a valuable protocol for large-scale production of FL and while conducive to further research and clinical use.1. Construction of pET30a-fll34Artificial and natural genes were used as templates to amplify the target fragment of FL gene, encoding the first 134 amino acid residues, by PCR. The target fragment was cloned into expression vector pET30a after introducing a Met residue in N-terminus and a Leu and Glu residue and His-tag in C-terminus, and then transformed into BL21 expression strain.2. Expression and purification of rhFL134BL21 expression strain was propagated in Lura broth. A series of methods, including low temperature induction, varied IPTG concentrations and ingredients of broth, shorten of induction time and combined aforementioned methods, fail to induce soluble rhFL134 expression or to induce natural gene fragment expression in any form. However, the target protein of artificial gene could be highly expressed as inclusion body and peaked at 6h after lmmol/L IPTG induction. Inclusion body was prepared by disrupting cell slurry using lysozyme and sonication, washed in order to enhance purity of target protein, and then denatured and refolded according to the optimal conditions determined by preliminary experiment. Soluble rhFL134 protein was loaded on HiTrap? affinity chromatography column after dialysis. SDS-PAGE and Western-blotting showed a single band of molecular mass of approximate 18 KD. The purity of rhFL134 is above 92%.3. Biological characterization of rhFL1343.1 rhFL134 promotes colony formation of mice marrow mononuclear cellsCompared with negative control rhFL134 can significantly promote formation of CFU-mix of mice marrow mononuclear cells in the context of mGM-CSF (20 ug/L) andmIL-3 (1 ml/L WEHI supernatant) . The effect is equivalent to that of commercial rhFL and relevant to the concentration of rhFL.3.2 rhFL134 promotes proliferation of CD34+progenitor cells of human cord blood in vitroMononuclear cells of human cord blood were separated and cultured in media containing different concentration of rhFL134 for one week. FCS analysis indicated 50 ug/L and 100 ug/L rhFL 134 could significantly promote proliferation of CD34+progenitor cells.To sum up, rhFL was highly expressed in E.Coli. BL21 strain by using artificial FL gene fragment as target: rhFL was successfully purified by a convenient one-step affinity chromatography; rhFL with high purity was acquired and proved to be high biological functioned. Our study is expected to provide a standard protocol for future large-scale production of the protein.