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Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins.

ABSTRACT: IPTG-inducible promoter is popularly used for the expression of recombinant proteins. However, it is not suitable at the industrial scale due to the high cost and toxicity on the producing cells. Recently, a Self-Inducible Expression (SILEX) system has developed to bypass such problems using Hsp70 as an autoinducer. Herein, the effect of other heat shock proteins on the autoinduction of green fluorescent protein (EGFP), romiplostim, and interleukin-2 was investigated. For quantitative measurements, EGFP expression was monitored after double-transformation of pET28a-EGFP and pET21a-(Hsp27/Hsp40/Hsp70) plasmids into E. coli using fluorimetry. Moreover, the expression level, bacterial growth curve, and plasmid and expression stability were compared to an IPTG- inducible system using EGFP. Statistical analysis revealed a significant difference in EGFP expression between autoinducible and IPTG-inducible systems. The expression level was higher in Hsp27 system than Hsp70/Hsp40 systems. However, the highest amount of expression was observed for the inducible system. IPTG-inducible and Hsp70 systems showed more lag-time in the bacterial growth curve than Hsp27/Hsp40 systems. A relatively stable EGFP expression was observed in SILEX systems after several freeze-thaw cycles within 90 days, while, IPTG-inducible system showed a decreasing trend compared to the newly transformed bacteria. Moreover, the inducible system showed more variation in the EGFP expression among different clones than clones obtained by SILEX systems. All designed SILEX systems successfully self-induced the expression of protein models. In conclusion, Hsp27 system could be considered as a suitable autoinducible system for protein expression due to less metabolic burden, lower variation in the expression level, suitable plasmid and expression stability, and a higher expression level.

SUBMITTER: Shariati FS

PROVIDER: S-EPMC7907268 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins.

Shariati Fatemeh Sadat FS Keramati Malihe M Valizadeh Vahideh V Cohan Reza Ahangari RA Norouzian Dariush D

Scientific reports 20210225 1

IPTG-inducible promoter is popularly used for the expression of recombinant proteins. However, it is not suitable at the industrial scale due to the high cost and toxicity on the producing cells. Recently, a Self-Inducible Expression (SILEX) system has developed to bypass such problems using Hsp70 as an autoinducer. Herein, the effect of other heat shock proteins on the autoinduction of green fluorescent protein (EGFP), romiplostim, and interleukin-2 was investigated. For quantitative measuremen ...[more]

PMID: 33633341

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Project description:Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaA A-04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaB A-04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaC A-04, 1770 bp), was identified. Sequence analysis of the phaA A-04, phaB A-04, and phaCA-04 genes revealed that phaC A-04 was 99% similar to phaC H16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaC A-04 was proline, whereas that of C. necator H16 was leucine. The intact phaCAB A-04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCAB A-04 and pColdTF-phaCAB A-04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a Y P/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCAB A-04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a Y P/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCAB A-04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

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Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins.

Publications

Comparison of E. coli based self-inducible expression systems containing different human heat shock proteins.

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