title: High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space.
abstract: A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.
authors.affiliation: Department of Application of Nuclear Techniques in Biological Sciences, IPEN-CNEN, Cidade Universitária, São Paulo, Brazil.
authors.forename: J E
authors.initials: JE
authors.lastname: de Oliveira
authors.forename: C R
authors.initials: CR
authors.lastname: Soares
authors.forename: C N
authors.initials: CN
authors.lastname: Peroni
authors.forename: E
authors.initials: E
authors.lastname: Gimbo
authors.forename: I M
authors.initials: IM
authors.lastname: Camargo
authors.forename: L
authors.initials: L
authors.lastname: Morganti
authors.forename: M H
authors.initials: MH
authors.lastname: Bellini
authors.forename: R
authors.initials: R
authors.lastname: Affonso
authors.forename: R R
authors.initials: RR
authors.lastname: Arkaten
authors.forename: P
authors.initials: P
authors.lastname: Bartolini
authors.forename: M T
authors.initials: MT
authors.lastname: Ribela
mesh_terms: D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D019382:Human Growth Hormone / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D019382:Human Growth Hormone