RESUMO
Human fibroblast growth factor 21 (hFGF21) has been characterized as an important regulator of glucose and lipid metabolism homeostasis. Here, to produce hFGF21 efficiently in Escherichia coli, the expression and solubility of hFGF21 were tested and optimised by fusing the protein with one of eight tags: hexahistidine (His6), thioredoxin (Trx), small ubiquitin-related modifier (Sumo), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilisation substance protein A (NusA), human protein disulphide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'). Each tag increased solubility of the protein when the expression temperature was 18°C. Unlike many other tags that were tested, MBP significantly enhanced the solubility of the protein also in the culture condition at 37°C. Thus, the MBP-hFGF21 construct was further pursued for optimisation of affinity chromatography purification. After tag removal, 8.1 mg of pure hFGF21 was obtained as a final product from 500 mL of starting culture. The protein was then characterised by mass spectroscopy and an in vitro functional assay using NIH-3T3 cells transfected with a ß-klotho reporter gene. These characteristics are similar to those of commercial hFGF21. Thus, the MBP tag is useful for efficient prokaryotic production and purification of bioactive hFGF21.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Escherichia coli/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Humanos , Proteínas Ligantes de Maltose/genética , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Human growth hormone (hGH) is synthesized by somatotroph cells of the anterior pituitary gland and induces cell proliferation and growth. This protein has been approved for the treatment of various conditions, including hGH deficiency, chronic renal failure, and Turner syndrome. Efficient production of hGH in Escherichia coli (E. coli) has proven difficult because the E. coli-expressed hormone tends to aggregate and form inclusion bodies, resulting in poor solubility. In this study, seven N-terminal fusion partners, hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), protein disulfide bond isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), were tested for soluble overexpression of codon-optimized hGH in E. coli. We found that MBP and hPDI tags significantly increased the solubility of the hormone. In addition, lowering the expression temperature to 18°C also dramatically increased the solubility of all the fusion proteins. We purified hGH from MBP-, PDIb'a'-, or Trx-tagged hGH expressed at 18°C in E. coli using simple chromatographic techniques and compared the final purity, yield, and activity of hGH to assess the impact of each partner protein. Purified hGH was highly pure on silver-stained gel and contained very low levels of endotoxin. On average, â¼37 mg, â¼12 mg, and â¼7 mg of hGH were obtained from 500 mL-cell cultures of Trx-hGH, MBP-hGH, and PDIb'a'-hGH, respectively. Subsequently, hGH was analyzed using mass spectroscopy to confirm the presence of two intra-molecular disulfide bonds. The bioactivity of purified hGHs was demonstrated using Nb2-11 cell.
Assuntos
Hormônio do Crescimento Humano/isolamento & purificação , Proteínas Ligantes de Maltose/metabolismo , Células Procarióticas/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Escherichia coli/metabolismo , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/farmacologia , Humanos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Células Procarióticas/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Human leukemia inhibitory factor (hLIF) is a multifunctional cytokine that is essential for maintaining the pluripotency of embryonic stem cells. hLIF may be also be useful in aiding fertility through its effects on increasing the implantation rate of fertilized eggs. Thus these applications in biomedical research and clinical medicine create a high demand for bioactive hLIF. However, production of active hLIF is problematic since eukaryotic cells demonstrate limited expression and prokaryotic cells produce insoluble protein. Here, we have adopted a hybrid protein disulfide isomerase design to increase the solubility of hLIF in Escherichia coli. Low temperature expression of hLIF fused to the b'a' domain of protein disulfide isomerase (PDIb'a') increased the soluble expression in comparison to controls. A simple purification protocol for bioactive hLIF was established that includes removal of the PDIb'a' domain by cleavage by TEV protease. The resulting hLIF, which contains one extra glycine residue at the N-terminus, was highly pure and demonstrated endotoxin levels below 0.05 EU/µg. The presence of an intramolecular disulfide bond was identified using mass spectroscopy. This purified hLIF effectively maintained the pluripotency of a murine embryonic stem cell line. Thus we have developed an effective method to produce a pure bioactive version of hLIF in E. coli for use in biomedical research.