Dataset Information

Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in Escherichia coli for facile production.

ABSTRACT: Prokaryotic expression systems are widely used to produce many types of biologics because of their extreme conveniences and unmatchable cost. However, production of recombinant human ferritin light chain (rhFTL) protein is largely restrained because its expression in Escherichia coli tends to form inclusion bodies (IBs). In this study, a prokaryotic expression vector (FTL-pBV220) harboring the rhFTL gene was constructed using a pBV220 plasmid. The tag-free rhFTL was highly expressed and almost entirely converted to soluble form, and thus the rhFTL was successfully self-assembled into uniform nanoparticles in E. coli. To establish a simplified downstream process, a precipitation procedure based on the optimized incubation temperature, pH condition, and ionic strength was developed to remove impurities from the crude lysate supernatant. The rhFTL retained in the clarified supernatant was subsequently purified in a single step using Capto Butyl column resulting in a considerable recovery and high purity. The purified rhFTL was characterized and verified by mass spectrometry and spectral and morphological analyses. The results revealed that rhFTL exhibited highly ordered and fairly compact structures and the spherical structures were preserved.

SUBMITTER: Song X

PROVIDER: S-EPMC9162929 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in <i>Escherichia coli</i> for facile production.

Song Xiaotong X Zheng Yongxiang Y Liu Yongdong Y Meng Huan H Yu Rong R Zhang Chun C

Engineering in life sciences 20220423 6

Prokaryotic expression systems are widely used to produce many types of biologics because of their extreme conveniences and unmatchable cost. However, production of recombinant human ferritin light chain (rhFTL) protein is largely restrained because its expression in <i>Escherichia coli</i> tends to form inclusion bodies (IBs). In this study, a prokaryotic expression vector (FTL-pBV220) harboring the rhFTL gene was constructed using a pBV220 plasmid. The tag-free rhFTL was highly expressed and a ...[more]

PMID: 35663479

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Project description:BACKGROUND: Bacterial inclusion bodies (IBs) are key intermediates for protein production. Their quality affects the refolding yield and further purification. Recent functional and structural studies have revealed that IBs are not dead-end aggregates but undergo dynamic changes, including aggregation, refunctionalization of the protein and proteolysis. Both, aggregation of the folding intermediates and turnover of IBs are influenced by the cellular situation and a number of well-studied chaperones and proteases are included. IBs mostly contain only minor impurities and are relatively homogenous. RESULTS: IBs of alpha-glucosidase of Saccharomyces cerevisiae after overproduction in Escherichia coli contain a large amount of (at least 12 different) major product fragments, as revealed by two-dimensional polyacrylamide gel electrophoresis (2D PAGE). Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight Mass-Spectrometry (MALDI-ToF MS) identification showed that these fragments contain either the N- or the C-terminus of the protein, therefore indicate that these IBs are at least partially created by proteolytic action. Expression of alpha-glucosidase in single knockout mutants for the major proteases ClpP, Lon, OmpT and FtsH which are known to be involved in the heat shock like response to production of recombinant proteins or to the degradation of IB proteins, clpP, lon, ompT, and ftsH did not influence the fragment pattern or the composition of the IBs. The quality of the IBs was also not influenced by the sampling time, cultivation medium (complex and mineral salt medium), production strategy (shake flask, fed-batch fermentation process), production strength (T5-lac or T7 promoter), strain background (K-12 or BL21), or addition of different protease inhibitors during IB preparation. CONCLUSIONS: alpha-glucosidase is fragmented before aggregation, but neither by proteolytic action on the IBs by the common major proteases, nor during downstream IB preparation. Different fragments co-aggregate in the process of IB formation together with the full-length product. Other intracellular proteases than ClpP or Lon must be responsible for fragmentation. Reaggregation of protease-stable alpha-glucosidase fragments during in situ disintegration of the existing IBs does not seem to occur.

Dataset Information

Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in Escherichia coli for facile production.

Publications

Conversion of recombinant human ferritin light chain inclusion bodies into uniform nanoparticles in <i>Escherichia coli</i> for facile production.

Similar Datasets

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