Enfuvirtide biosynthesis in thermostable chaperone-based fusion

Zenin, Vladimir A.; Yurkova, Maria S.; Tsedilin, Andrey M.; Федоров, А. Н.

doi:10.1016/j.btre.2022.e00734

Cited by 2 publications

(6 citation statements)

References 38 publications

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“…The SDS-PAGE of analytical 0–5 h hydrolysis in formic acid was previously published with no visible improvement in process yield after the first hour [ 3 ].…”

Section: Resultsmentioning

confidence: 99%

“…Modified GroEL-Enfuvirtide fusion was produced as described previously [ 3 ] The protein with the designed sequence ( S1 File ) was expressed in E . coli cell culture in soluble form, purified by heat-induced host protein denaturation and IEX chromatography, desalted, lyophilized, and stored at -20°C.…”

Section: Methodsmentioning

confidence: 99%

“…HPLC-MS/MS analysis was performed as described earlier [ 3 ]. Impact II QqTOF high-resolution mass-spectrometer (Bruker Daltonik, Germany) equipped with Elute UHPLC (Bruker Daltonik, Germany) and Intensity Solo 1.8 C18-2 2.1*100 mm 1.8 μm 90 Å reverse-phase column (Bruker Daltonik, Germany) was used.…”

Section: Methodsmentioning

confidence: 99%

“…SDS-PAGE in tris-glycine buffer and 10–20% gradient gel was performed as previously described [ 3 ].…”

Section: Methodsmentioning

confidence: 99%

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Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities

Zenin¹,

Tsedilin²,

Yurkova³

et al. 2023

PLoS ONE

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Large peptide biosynthesis is a valuable alternative to conventional chemical synthesis. Enfuvirtide, the largest therapeutic peptide used in HIV infection treatment, was synthesized in our thermostable chaperone-based peptide biosynthesis system and evaluated for peptide quality as well as the profile of process-related impurities. Host cell proteins (HCPs) and BrCN cleavage-modified peptides were evaluated by LC-MS in intermediate. Cleavage modifications during the reaction were assessed after LC-MS maps were aligned by simple in-house algorithm and formylation/oxidation levels were estimated. Circular dichroism spectra of the obtained enfuvirtide were compared to the those of the chemically- synthesized standard product. Final-product endotoxin and HCPs content were assessed resulting 1.06 EU/mg and 5.58 ppm respectively. Peptide therapeutic activity was measured using the MT-4 cells HIV infection-inhibition model. The biosynthetic peptide IC50 was 0.0453 μM while the standard one had 0.0180 μM. Non-acylated C-terminus was proposed as a cause of IC50 and CD spectra difference. Otherwise, the peptide has met all the requirements of the original chemically synthesized enfuvirtide in the cell-culture and in vivo experiments.

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“…The SDS-PAGE of analytical 0–5 h hydrolysis in formic acid was previously published with no visible improvement in process yield after the first hour [ 3 ].…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…SDS-PAGE in tris-glycine buffer and 10–20% gradient gel was performed as previously described [ 3 ].…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities

Zenin¹,

Tsedilin²,

Yurkova³

et al. 2023

PLoS ONE

Self Cite

View full text Add to dashboard Cite

show abstract

“…The peptide was inserted into the GroEL polypeptide chain in such a way that it did not interfere with the formation of the correct GroEL oligomeric structure, but protruded into the oligomer cavity and interacted with the substrate-binding surface. For this purpose, a methionineless variant of thermostable chaperon GroEL from Thermus thermophilus was designed [ 27 ], and two target peptides—a toxic antimicrobial polypeptide polyphemusin I [ 22 ] and a therapeutic peptide for the treatment of AIDS patients enfuvirtide [ 28 ]—were used. In both cases, the polypeptide fused with GroEL was expressed with high yield in a soluble state, and the target polypeptide was separated from GroEL by CNBr cleavage.…”

Section: Introductionmentioning

confidence: 99%

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties

et al. 2023

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The incorporation of photoproteins into proteins of interest allows the study of either their localization or intermolecular interactions in the cell. Here we demonstrate the possibility of in vivo incorporating the photoprotein Aequorea victoria enhanced green fluorescent protein (EGFP) or Gaussia princeps luciferase (GLuc) into the tetradecameric quaternary structure of GroEL chaperonin and describe some physicochemical properties of the labeled chaperonin. Using size-exclusion and affinity chromatography, electrophoresis, fluorescent and electron transmission microscopy (ETM), small-angle X-ray scattering (SAXS), and bioluminescence resonance energy transfer (BRET), we show the following: (i) The GroEL14-EGFP is evenly distributed within normally divided E. coli cells, while gigantic undivided cells are characterized by the uneven distribution of the labeled GroEL14 which is mainly localized close to the cellular periplasm; (ii) EGFP and likely GLuc are located within the inner cavity of one of the two GroEL chaperonin rings and do not essentially influence the protein oligomeric structure; (iii) GroEL14 containing either EGFP or GLuc is capable of interacting with non-native proteins and the cochaperonin GroES.

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Enfuvirtide biosynthesis in thermostable chaperone-based fusion

Cited by 2 publications

References 38 publications

Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities

Thermostable chaperone-based polypeptide biosynthesis: Enfuvirtide model product quality and protocol-related impurities

In Vivo Incorporation of Photoproteins into GroEL Chaperonin Retaining Major Structural and Functional Properties

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