Twin arginine translocation system in secretory expression of recombinant human growth hormone

Abstract

Recombinant protein production in E. coli has several advantages over other expression systems. Misfolding, inclusion body formation, and lack of eukaryotic post translational modification are the most disadvantages of this system. Exporting of correctly folded proteins to the outside of reductive cytoplasmic environment through twin-arginine system could help to pass these limiting steps. Two signal sequences, TorA and SufI are used at N-terminal of human growth hormone (hGH) bearing DsbA gene sequence at C-terminal to enhance folding. The synthetic cassettes including the signal sequence, hGH and DsbA were transformed into E. coli BL21 (DE3) to study the effect of signal sequence and DsbA chaperone on translocation and folding of the protein. The results confirmed using signal sequence at N-terminal of targeted protein and coexpression with DsbA could transport proteins to the periplasmic space and culture media compared to control groups. Although there is no protein band of somatropin in SDS-Page of culture media samples when using SufI as signaling sequence, the study demonstrated TorA signal sequence could transport the target protein to the culture media. However, there was a considerable amount of hGH in periplasmic space when using SufI compared to control.

Keywords: DsbA; Growth hormone; Signal sequence; SufI; TorA; Twin arginine translocation.

Fig. 1

pET-22b(+) was used for expression of target genes. Cassettes including the signal sequence, somatropin and DsbA were synthesized and ligated to pET-22b(+) using appropriate endonuclease. Signal sequence TorA was used for cassette I, NdeI and NcoI restriction sites were added to the ends of the cassette for digestion/ligation processes. Cassette III was synthesized by the SufI signal sequence and NdeI and XhoI restriction sites were used at the ends for manipulation. Enterokinase site was also added betweeen the signal sequence and somatropin. Ribosome binding site was used between the two gene sequences (somatropin and DsbA).

Fig. 2

The size of synthetic cassettes were cloned to E. coli DH5α. Following plasmid preparation, digestions were carried out with appropriate endonucleases (NdeI and NcoI for Cassette I and NdeI and XhoI for cassette III). The cassette sizes were 1465 and 1426 bp for cassette I and III, respectively. (L) DNA Ladder Mix; (I) recombinant pGEM-Cassette I digested with NdeI and NcoI; (II) recombinant pGEM-Cassette I digested with EcoR1; (III) recombinant pGEM-Cassette III digested with EcoR1; and (IV) recombinant pGEMCassette III digested with NdeI and XhoI.

Fig. 3

The expression pattern of somatropin using TorA as signaling sequence/DsbA. (L) protein molecular weight marker; (I) secretory fraction to culture media; (II) cytoplasmic fraction; (III) periplasmic fraction following osmotic shock with a hypertonic solution; (IV) periplasmic fraction following osmotic shock with a hypotonic solution. The figure shows although the target protein is exported to periplasmic space and outside the cell, most of the protein is still located in cytoplasm. The hypertonic solution is eluted more protein compared to the hypotonic solution. The molecular weight of somatropin is 22 kDa. The band around 45 kDa is the dimer form of target protein according to the western blotting result.

Fig. 4

The expression pattern of the control group. The cassette containing somatropin without signaling sequence/DsbA. The periplasmic solutions were pooled and loaded on the gel as one fraction. The target protein, somatropin, with molecular weigh 22 kDa was not seen in periplasmic and culture media fractions (Lane II and III). All the expressed proteins were accumulated in the cytoplasm. Although expression system is capable of producing proteins in the absence of signaling sequence/DsbA (Lane I), the proteins form inclusion body in the cytoplasm. (L) protein molecular weight marker; (I) cytoplasmic fraction; (II) periplasmic fraction following the osmotic shock; (III) secretory fraction to culture media. The band around 45 kDa is the dimer form of target protein.

Fig. 5

The expression pattern of somatropin using SufI as signaling sequence and coexpression with DsbA as chaperone. (L) protein molecular weight marker; (I) secretory fraction to culture media; (II) periplasmic fraction following osmotic shock with a hypertonic solution; (III) periplasmic fraction following osmotic shock with a hypotonic solution; (IV) cytoplasmic fraction. Although, there is a nice and sharp band regarding the somatropin in periplasmic solution (lane II), there are much proteins is still located in the cytoplasm. Exporting the protein outside the cell was not successful using SufI as signaling sequence. The band around 45 kDa is the dimer form of target protein according to the western blotting result.

Fig. 6

Western blot of the expression pattern of somatropin when using TorA/DsbA. (L) protein molecular weight marker; (I) cytoplasmic fraction; (II) secretory fraction to culture media; (III) periplasmic fraction following osmotic shock with a hypertonic solution; (IV) periplasmic fraction following osmotic shock with a hypotonic solution. The figure shows although the target protein is exported to periplasmic space and outside the cell, most of the protein is still located in cytoplasm. The hypertonic solution is eluted more protein compared to the hypotonic solution. The molecular weight of somatropin is 22 kDa. The band around 45 kDa is the dimer form.