Optimization of expression, purification and secretion of functional recombinant human growth hormone in Escherichia coli using modified staphylococcal protein a signal peptide

Abstract

Background Human Growth Hormone (hGH) is a glycoprotein released from the pituitary gland. Due to the wide range of effects in humans, any disruption in hGH secretion could have serious consequences. This highlights the clinical importance of hGH production in the treatment of different diseases associated with a deficiency of this hormone. The production of recombinant mature hormone in suitable hosts and secretion of this therapeutic protein into the extracellular space can be considered as one of the best cost-effective approaches not only to obtain the active form of the protein but also endotoxin-free preparation. Since the natural growth hormone signal peptide is of eukaryotic origin and is not detectable by any of the Escherichia coli secretory systems, including Sec and Tat, and is therefore unable to secrete hGH in the prokaryotic systems, designing a new and efficient signal peptide is essential to direct hGh to the extracellular space. Results In this study, using a combination of the bioinformatics design and molecular genetics, the protein A signal peptide from Staphylococcus aureus was modified, redesigned and then fused to the mature hGH coding region. The recombinant hGH was then expressed in E. coli and successfully secreted to the medium through the Sec pathway. Secretion of the hGH into the medium was verified using SDS-PAGE and western blot analysis. Recombinant hGH was then expressed in E. coli and successfully secreted into cell culture medium via the Sec pathway. The secretion of hGH into the extracellular medium was confirmed by SDS-PAGE and Western blot analysis. Furthermore, the addition of glycine was shown to improve hGH secretion onto the culture medium. Equations for determining the optimal conditions were also determined. Functional hGH analysis using an ELISA-based method confirmed that the ratio of the active form of secreted hGH to the inactive form in the periplasm is higher than this ratio in the cytoplasm. Conclusions Since the native signal protein peptide of S. aureus protein A was not able to deliver hGH to the extracellular space, it was modified using bioinformatics tools and fused to the n-terminal region of hGh to show that the redesigned signal peptide was functional.

Growth hormone receptor agonists and antagonists: From protein expression and purification to long‐acting formulations

“…In a recent report, a modified Staphylococcus aureus protein A signal peptide fused to the mature hGH coding region was utilized that allowed rhGH to be secreted through the Sec pathway. This increased expression 3-fold compared with cytoplasmic expression (Rigi et al, 2021). In addition, Perez-Perez et al (2020) developed a novel expression method using a signal peptide from PelB fused to small metal-binding protein (PelB-SmbP) that combines the Anion-exchange 800 mg hGHA1 and 1.2 g hGHA2 from 200 L culture (Li et al, 2004) Abbreviations: GH, growth hormone; GHA, GH receptor antagonist; GHA1, Cys-hGH-del1-4, G120R, K168A, E174A, C182S, del186-191; GHA2, hGH-H21A, G120R, E174A; hGH, human growth hormone; IMAC, immobilized-metal affinity chromatography; N/A, not available.…”

“…The signal peptide is cleaved by a signal peptidase during or shortly after substrate translocation, and the mature protein is released on the trans-side of the membrane (Freudl, 2018). Periplasmic secretion has been widely used for the production of rhGH (Alanen et al, 2015;Amaranto et al, 2021;Bagherinejad et al, 2016Bagherinejad et al, , 2018Becker & Hsiung, 1986;Browning et al, 2017;Chang et al, 1987;Chang et al, 1989;Ghorpade & Garg, 1993;Gray et al, 1985;Guerrero Montero et al, 2019;Jeiranikhameneh et al, 2017;Li et al, 2004;Matos et al, 2014;Menezes et al, 2017;Perez-Perez et al, 2020;Rigi et al, 2021;Soares et al, 2003;Soares et al, 2008;Sockolosky & Szoka, 2013;Teresa et al, 2000;Uchida et al, 1997;Zamani et al, 2016;Zhou et al, 2021). A common approach is to fuse the signal sequence to the N-terminus of rhGH for transport, and a His-tag to the C-terminus for subsequent purification.…”

“…In a recent report, a modified Staphylococcus aureus protein A signal peptide fused to the mature hGH coding region was utilized that allowed rhGH to be secreted through the Sec pathway. This increased expression 3‐fold compared with cytoplasmic expression (Rigi et al, 2021 ). In addition, Perez‐Perez et al ( 2020 ) developed a novel expression method using a signal peptide from PelB fused to small metal‐binding protein (PelB‐SmbP) that combines the benefits of periplasmic expression with purification via immobilized Ni affinity chromatography.…”

Secretion and Periplasmic Activation of a Potent Endonuclease in E. coli

“…Considering the potential toxicity of cytoplasmically accumulated SmEn and the requirement to form two disulfide bonds for activity and stability [10], we aimed to translocate SmEn to the periplasm of E. coli, where DsbA and DsbC could assist oxidative protein folding [11]. Secretion of proteins into the periplasmic space followed by osmotic extraction also potentially reduces contamination of product proteins with endotoxins and native E. coli proteins to reduce downstream processing costs compared to cytoplasmic expression [12]. Periplasmic accumulated may also improve folding because of lower protein-protein interactions compared to cytoplasmic expression, and the addition of an N-terminal methionine is avoided [13, 14].…”

PERISCOPE-Opt: Machine learning-based prediction of optimal fermentation conditions and yields of recombinant periplasmic protein expressed in Escherichia coli

“…The periplasmic expression of recombinant proteins is preferred because the periplasmic space provides an oxidized environment that improves protein folding, especifically for proteins containing di-sulphide bonds. Moreover, the target proteins can be selectively recovered from the periplasmic space using the milder cell disruption steps that avoid the release of cytoplasmic content to the processing fluid [5] , [6] , [7] , [8] . The increasing demands for recombinant proteins have driven the necessity of optimizing various fermentation process parameters to achieve the maximal RPP.…”