Functional expression, purification, and characterization of human Flt3 ligand in the Pichia pastoris system

Chi

Biotechnology & Biotechnological Equipment

et al. 2014

MAP30, a single-stranded type-I ribosome inactivating protein found in Momordica charantia, shows anti-HIV and anti-tumour activity. It could significantly inhibit the HIV-1 and herpes simplex virus infection. In this study, we tried a safe and convenient expression system supplying MAP30 protein for medical practice. The gene encoding MAP30 was cloned into pMD18-T vector. The pMD18-MAP30 plasmid was transformed into competent Escherichia coli JM109 by a chemical method. The MAP30 gene was obtained from the pMD18-MAP30 plasmid digested with NotI and SnaBI and the MAP30 gene was ligated into pGAPHα. Then, pGAPHα-MAP30 was transformed into Pichia pastoris GS115 by electroporation. GS115 transformants were analysed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and Western blot. SDS-PAGE revealed an extra band of approximately 32 kDa in the supernatant protein of the GS115 transformants and in their intracellular protein fraction. The result of Western-blot analysis showed that the supernatant and the cell pellet from GS115 with pGAPHα-MAP30 could specially bind to monoclonal antibodies against His in the 32 kDa site. These results demonstrated that the expression of MAP30 in P. pastoris was successful; the process of the expression did not need methanol induction or introduction of an antibiotic-resistance gene. The study may provide a new way for MAP30 synthesis. Owing to its safety, this new approach is expected to be widely used in the medical field.

Section: Introductionmentioning

confidence: 99%

Gene cloning and expression of MAP30 inPichia pastoris

Chi

Biotechnology & Biotechnological Equipment

et al. 2014

“…The FLT3L induced expansion of the cells result in NKDC with a more mature phenotype and a slightly increased ability to capture and process antigen (Zhang et al, 2009). Overall, FLT3L has been found to be the most potent growth factor for DC generation both in vitro and in vivo, resulting in its continued research in immunotherapy due to its ability to present tumor antigens to T-cells and irradiate tumors (Zhang et al, 2005).…”

Section: Biological Significance Of Flt3mentioning

confidence: 99%

“…In 2005, Zhang et al cloned FLT3L in the Pichia pastoris (P. pastoris) system and were able to get a yield of 30 mg/L of recombinant FLT3L with a purity of 95% utilizing dialysis, filtration and anion-exchange steps. A synthetic DNA fragment was constructed based on P. pastoris biased codon usage encoding soluble human FLT3L, supernatant was analysed for expression of FLT3L and dialyzed against 20 mM Tris-Cl pH 8.0 with a molecular weight cut off of 12kDa and filtered through a 0.45um filter (Zhang, 2005). After application to an anionexchange column, bound protein was eluted with a linear salt gradient (Zhang, 2005).…”

Section: S=!mentioning

confidence: 99%

Cloning, expression and purification of the cytokine FLT3-ligand for protein interaction studies

Agro¹

2021

Preprint

The FLT3-FLT3L receptor complex is responsible for the proliferation and differentiation of hematopoietic progenitor and stem cells, and is involved in several hematological malignancies. A truncated, soluble form of FLT3L was amplified from the full-length sequence by PCR and cloned into a genomic integration and expression vector for Kluyveromyces lactis. Expression and secretion of the 161 amino acid FLT3L was analyzed by SDS-PAGE and Western blot and showed smeared bands around 50kDa consistent with a high level of varying glycosylation as expected. A yield of 100ug/mL of culture was obtained. Purification was performed by Ni-affinity chromatography and the protein was confirmed to be in the elution fraction via mass spectrometry analysis. Purified FLT3L was able to activate RAW264.7 cells after 24hr incubation period causing large vacuoles and a dendritic appearance. FLT3L will be utilized for LARC protein interaction studies to search for interactors that might serve as therapeutic agents in human body fluids and cell culture models of myeloid leukemia.

“…Soluble FL has three disulfide bond knots, which make it difficult to directly express FL as a soluble and bioactive protein in the cytoplasm of E. coli. Recombinant hFL has been expressed in yeast as a secretory protein [23] and in E. coli as an inclusion body [24]. Here, we expressed soluble rhFL in the periplasm of E. coli.…”

Section: Introductionmentioning

confidence: 99%

Expression of a recombinant FLT3 ligand and its emtansine conjugate as a therapeutic candidate against acute myeloid leukemia cells with FLT3 expression

et al. 2021

Background Most patients with acute myeloid leukemia (AML) remain uncurable and require novel therapeutic methods. Gain-of-function FMS-like tyrosine kinase 3 (FLT3) mutations are present in 30–40% of AML patients and serve as an attractive therapeutic target. In addition, FLT3 is aberrantly expressed on blasts in > 90% of patients with AML, making the FLT3 ligand-based drug conjugate a promising therapeutic strategy for the treatment of patients with AML. Here, E. coli was used as a host to express recombinant human FLT3 ligand (rhFL), which was used as a specific vehicle to deliver cytotoxic drugs to FLT3 + AML cells. Methods Recombinant hFL was expressed and purified from induced recombinant BL21 (DE3) E. coli. Purified rhFL and emtansine (DM1) were conjugated by an N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) linker. We evaluated the potency of the conjugation product FL-DM1 against FLT3-expressing AML cells by examining viability, apoptosis and the cell cycle. The activation of proteins related to the activation of FLT3 signaling and apoptosis pathways was detected by immunoblotting. The selectivity of FL-DM1 was assessed in our unique HCD-57 cell line, which was transformed with the FLT3 internal tandem duplication mutant (FLT3-ITD). Results Soluble rhFL was successfully expressed in the periplasm of recombinant E. coli. The purified rhFL was bioactive in stimulating FLT3 signaling in AML cells, and the drug conjugate FL-DM1 showed activity in cell signaling and internalization. FL-DM1 was effective in inhibiting the survival of FLT3-expressing THP-1 and MV-4-11 AML cells, with half maximal inhibitory concentration (IC50) of 12.9 nM and 1.1 nM. Additionally, FL-DM1 induced caspase-3-dependent apoptosis and arrested the cell cycle at the G2/M phase. Moreover, FL-DM1 selectively targeted HCD-57 cells transformed by FLT3-ITD but not parental HCD-57 cells without FLT3 expression. FL-DM1 can also induce obvious apoptosis in primary FLT3-positive AML cells ex vivo. Conclusions Our data demonstrated that soluble rhFL can be produced in a bioactive form in the periplasm of recombinant E. coli. FL can be used as a specific vehicle to deliver DM1 into FLT3-expressing AML cells. FL-DM1 exhibited cytotoxicity in FLT3-expressing AML cell lines and primary AML cells. FL-DM1 may have potential clinical applications in treating patients with FLT3-positive AML.