Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis

Silva, Felipe D.; Freire, Renan Passos; Suzuki, Miriam F.; Soares, Carlos R.J.; Bartolini, Paolo

doi:10.1186/s13568-019-0856-8

Cited by 8 publications

(6 citation statements)

References 35 publications

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“…In both cases, the purification was carried out using an ÄKTA purification system (GE Health Sciences, Buckinghamshire, UK) (Menezes et al 2017 ; Silva et al 2019 ).…”

Section: Methodsmentioning

confidence: 99%

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Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

et al. 2021

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The human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

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“…In both cases, the purification was carried out using an ÄKTA purification system (GE Health Sciences, Buckinghamshire, UK) (Menezes et al 2017 ; Silva et al 2019 ).…”

Section: Methodsmentioning

confidence: 99%

“…The mixture was applied on a GroundSteel plate and analyzed between 10 and 30 kDa. The acquisition mode was linear with positive polarity (Protparam program) (Silva et al 2019 ).…”

Section: Methodsmentioning

confidence: 99%

Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

et al. 2021

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“…The mixture was applied on a GroundSteel plate and analyzed between 10 kDa and 30 kDa. The acquisition mode was linear with positive polarity (Protparam program) (Silva et al 2019).…”

Section: Hpsec and Mass Spectral Characterizationmentioning

confidence: 99%

“…In both cases, the puri cation was carried out using an ÄKTA puri cation system (GE Health Sciences, Buckinghamshire, UK) (Menezes et al 2017;Silva et al 2019).…”

Section: Puri Cation Processmentioning

confidence: 99%

Periplasmic Synthesis and Purification of the Human Prolactin Antagonist Δ1-11-G129R-hPRL

Almeida

Pomin

Silva

et al. 2021

Preprint

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The human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b(+) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157±0.015 μg/mL/A600 at a final optical density of 3.43±0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF-MS. The final product presented >95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

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“…On the other hand, overexpression in mammalian systems offers molecular chaperones and co-factors that allow for the production of correctly folded proteins with the desired PTMs, which are important for their functionality and full activity [ 30 , 31 ]. So far, most reports describe the purification of glycosylated proteins [ 32 , 33 , 34 ]; however, a detailed protocol describing the step-by-step overexpression and purification of palmitoylated recombinant proteins is currently missing.…”

Section: Introductionmentioning

confidence: 99%

High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

et al. 2021

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Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein–protein or protein–membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

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Expression of glycosylated human prolactin in HEK293 cells and related N-glycan composition analysis

Cited by 8 publications

References 35 publications

Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

Periplasmic Synthesis and Purification of the Human Prolactin Antagonist Δ1-11-G129R-hPRL

High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells

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