High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space

Oliveira, João Ezequiel; Soares, Carlos R.J.; Peroni, Cibele N.; Gimbo, Elizabeth; Camargo, Iara Maria Carneiro de; Morganti, Lígia; Bellini, Maria Helena; Affonso, Regina; Arkaten, Rosangela; Bartolini, Paolo; Ribela, M.T.C.P.

doi:10.1016/s0021-9673(99)00613-5

Cited by 46 publications

(22 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Alternative expression systems have been based on the secretion of the protein into the E. coli periplasmic space, which not only allows a greater chance to obtain the protein in a folded and soluble form but also lower load of contaminating proteins in the periplasmic fluid makes purification process easier. Secretion process in the periplasmic space of E. coli cells mimics the natural process of somatotropic cells in the pituitary gland and has been achieved by linking the signal peptide sequence to the human GH (de Oliveira et al 1999;Teresa et al 2000;Soares et al 2003).…”

Section: Introductionmentioning

confidence: 99%

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane

Durrani

Gul

Sadaf

et al. 2015

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

This study shows expression of recombinant ovine growth hormone (roGH) and targeting to the inner membrane using signal sequence, DsbA, in Escherichia coli (E. coli) cell. Factors such as temperature, IPTG induction, and expression conditions were studied and show diverse optical density with different media compositions. The optimum expression level of roGH in terrific broth medium was at 25 °C on induction with 20 μM IPTG in early logarithmic phase. SDS-PAGE analysis of expression and subcellular fractions of recombinant constructs revealed the translocation of roGH to the inner membrane of E. coli with DsbA signal sequence at the N terminus of roGH. The protein was easily solubilized by 40 % acetonitrile with ~90 % purity and was identified by Western blot, and analysis on MALDI-TOF/TOF confirmed a size of 21,059 Da. Relatively high soluble protein yield of 65.3 mg/L of roGH was obtained. The biological function of roGH was confirmed by HeLa cell line proliferation. This is the first study describing achievement of biologically active soluble roGH targeted to the inner membrane of E. coli and rapid purification with high yield.

show abstract

Section: Introductionmentioning

confidence: 99%

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane

Durrani

Gul

Sadaf

et al. 2015

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

show abstract

“…Depending on the nature of the product and culture process, crude recombinant proteins are typically harvested in the late exponential or the early stationary phase of growth and are subjected to a multistep purification process to purify the recombinant product from the culture medium, which also contains contaminants such as host cell proteins and nucleic acids (2). During the developmental process, removal of CHO cell-derived proteins from the recombinant product is monitored using multiproduct immunoassays.…”

Section: Introductionmentioning

confidence: 99%

Proteomic Profiling of Secreted Proteins from CHO Cells Using Surface-Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry

et al. 2008

View full text Add to dashboard Cite

Chinese hamster ovary (CHO) cells are the most commonly used host cell line for the production of recombinant biopharmaceuticals. These biopharmaceuticals are typically secreted from CHO cells and purified from harvested cell culture media. The purpose of this study was to investigate changes in the secreted proteome of CHO cells over the various stages of the growth cycle using Surface Enhanced Laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Conditioned media samples were collected each day over a 6 day growth period from CHO-K1 cells grown in low serum (0.5% FBS) conditions in monolayer culture. Samples were profiled on a number of ProteinChip arrays with different chromatographic surfaces. From this study, 24 proteins were found to be differentially regulated at different phases of the growth cycle in CHO-K1 cells, when profiled on two chromatographic surfaces, Q10 (anionic) and IMAC30 (metal affinity) ProteinChip arrays.

show abstract

“…Secretion of the protein to the periplasmic space is also employed to produce human growth hormone (hGH) in E. coli [8]. There are numerous versions of this recombinant protein on the market, some produced in yeast or mammalian cells [4].…”

Section: Overcoming Missing Posttranslational Modificationsmentioning

confidence: 99%

Engineering of Therapeutic Proteins Production in Escherichia coli

Kamionka¹

2011

curr pharm biotechnol

144

View full text Add to dashboard Cite

Low cost and simplicity of cultivating bacteria make the E. coli expression system a preferable choice for production of therapeutic proteins both on a lab scale and in industry. In addition straightforward recombinant DNA technology offers engineering tools to produce protein molecules with modified features. The lack of posttranslational modification mechanisms in bacterial cells such as glycosylation, proteolytic protein maturation or limited capacity for formation of disulfide bridges may, to a certain extent, be overcome with protein engineering. Protein engineering is also often employed to improve protein stability or to modulate its biological action. More sophisticated modifications may be achieved by genetic fusions of two proteins. This article presents a variety of examples of genetic engineering of therapeutic proteins. It emphasizes the importance of designing a construct without any unnecessary amino acid residues.

show abstract

High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space

Cited by 46 publications

References 27 publications

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane

Expression and rapid purification of recombinant biologically active ovine growth hormone with DsbA targeting to Escherichia coli inner membrane

Proteomic Profiling of Secreted Proteins from CHO Cells Using Surface-Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry

Engineering of Therapeutic Proteins Production in Escherichia coli

Contact Info

Product

Resources

About