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Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon ?-2b.

ABSTRACT: Escherichia coli is an extensively used host for the production of recombinant proteins, making its N-terminal methionine aminopeptidase (MAP) an attractive candidate for studies on posttranslational protein processing. The present study describes the recombinant production and properties of MAP from the DH5? strain of E. coli. The soluble and active enzyme was produced in E. coli BL21 (DE3) RIL - codon plus cells under a T7 promoter system and purified by anion-exchange chromatography. It exhibited a molecular weight of 29,200.94 Da by MALDI-TOF analysis. The purified enzyme showed specific activity of 1.64 U/mg with methionylp-nitroanilide and 1.51 U/mg with synthetic tetrapeptide substrate 'MGMM' in a discontinuous HPLC-based assay. In vitro studies showed the processing of up to 36% of Met-INF?-2b in 40 min. In silico studies revealed that the ES-complex formation between the enzyme and interferon has a ?G -683.07 kJ/mol. Molecular docking results showed that the processed INF?-2b has greater binding affinity with IFNAR2 receptor as indicated by ?G -784.53 kJ/mol, significantly lower than that of methionine containing INF?-2b (?G -717.63 kJ/mol). These findings emphasize the functional superiority or better efficacy of N-terminal methionine processed recombinant interferon.

SUBMITTER: Arif A

PROVIDER: S-EPMC6353284 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon α-2b.

Arif Amina A Mohammed Kaleemuddin K Nadeem Muhammad Shahid MS

Turkish journal of biology = Turk biyoloji dergisi 20180613 3

Escherichia coli is an extensively used host for the production of recombinant proteins, making its N-terminal methionine aminopeptidase (MAP) an attractive candidate for studies on posttranslational protein processing. The present study describes the recombinant production and properties of MAP from the DH5α strain of E. coli. The soluble and active enzyme was produced in E. coli BL21 (DE3) RIL - codon plus cells under a T7 promoter system and purified by anion-exchange chromatography. It exhib ...[more]

PMID: 30814886

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Project description:BackgroundLike most protein macromolecular drugs, the half-life of rhIFNɑ-2b is short, with a low drug utilization rate, and the preparation and release conditions significantly affect its stability.MethodsA nanoporous ion-responsive targeted drug delivery system (PIRTDDS) was designed to improve drug availability of rhIFNα-2b and target it to the lung passively with sustained release. Chitosan rhIFNα-2b carboxymethyl nanoporous microspheres (CS-rhIFNα-2b-CCPM) were prepared by the column method. Here, an electrostatic self-assembly technique was undertaken to improve and sustain rhIFNα-2b release rate.ResultsThe size distribution of the microspheres was 5~15 μm, and the microspheres contained nanopores 300~400 nm in diameter. The in vitro release results showed that rhIFNα-2b and CCPM were mainly bound by ionic bonds. After self-assembling, the release mechanism was transformed into being membrane diffusion. The accumulative release amount for 24 hrs was 83.89%. Results from circular dichrogram and SDS-PAGE electrophoresis showed that there was no significant change in the secondary structure and purity of rhIFNα-2b. Results from inhibition rate experiments for A549 cell proliferation showed that the antitumor activity of CS-rhIFNα-2b-CCPM for 24 hrs retained 91.98% of the stock solution, which proved that the drug-loaded nanoporous microspheres maintained good drug activity. In vivo pharmacokinetic experimental results showed that the drugs in CS-rhIFNα-2b-CCPM can still be detected in vivo after 24 hrs, equivalent to the stock solution at 6 hrs, which indicated that CS-rhIFNα-2b-CCPM had a certain sustained-release effect in vivo. The results of in vivo tissue distribution showed that CS-rhIFNα-2b-CCPM was mainly concentrated in the lungs of mice (1.85 times the stock solution). The pharmacodynamics results showed that CS-rhIFNα-2b-CCPM had an obvious antitumor effect, and the tumor inhibition efficiency was 29.2%.ConclusionThe results suggested a novel sustained-release formulation with higher drug availability and better lung targeting from CS-rhIFNα-2b-CCPM compared to the reference (the stock solution of rhIFNα-2b), and, thus, should be further studied.

Dataset Information

Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon ?-2b.

Publications

Biochemical and in silico evaluation of recombinant E. coli aminopeptidase and in vitro processed human interferon α-2b.

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