Engineered small metal‐binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli
| Autor: | Jose Ruben Morones-Ramirez, Isaías Balderas-Rentería, Elizeth Pioquinto‐Avila, Xristo Zarate, David A. Perez-Perez, Eder Arredondo-Espinoza |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Signal peptide QH301-705.5 Recombinant Fusion Proteins Nitrosomonas europaea SmbP Protein tag Protein Sorting Signals medicine.disease_cause General Biochemistry Genetics and Molecular Biology 03 medical and health sciences 0302 clinical medicine Bacterial Proteins Affinity chromatography Metalloproteins Escherichia coli medicine Humans Biology (General) Research Articles Polysaccharide-Lyases periplasm PelB‐SmbP Chemistry Periplasmic space Fusion protein Transport protein Protein Transport 030104 developmental biology Metabolic Engineering Biochemistry 030220 oncology & carcinogenesis human growth hormone Target protein Carrier Proteins Research Article protein expression and purification |
| Zdroj: | FEBS Open Bio, Vol 10, Iss 4, Pp 546-551 (2020) FEBS Open Bio |
| ISSN: | 2211-5463 |
| Popis: | Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C‐terminal his‐tag added for subsequent purification. Our research group has proposed the small metal‐binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB‐SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2‐11 cells, was equivalent to commercial growth hormone at 50 ng·mL−1. Therefore, we strongly recommend the use of PelB‐SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli. This work describes the production of bioactive human growth hormone tagged with the fusion protein PelB‐SmbP in the periplasm of Escherichia coli. Here, we report the highest periplasmic production for this protein so far. Therefore, PelB‐SmbP is an attractive tag for the expression and purification of other target therapeutic proteins in Escherichia coli. |
| Databáze: | OpenAIRE |
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