Large–Scale Affinity Purification of Human Insulin–Like Growth Factor I from Culture Medium of Escherichia Coli

Moks, Tomas; Abrahmsén, Lars; Österlöf, Björn; Josephson, Staffan; Östling, Martin; Enfors, Sven‐Olof; Persson, Ingalill; Nilsson, Björn; Uhlén, Mathias

doi:10.1038/nbt0487-379

Cited by 117 publications

(25 citation statements)

References 9 publications

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“…The sequence ASGR links the protein A sequence to the amino acids 1-202 of LexA (J01643), which in turn is fused to EFPGIW and the amino acids 402-479 of VP16 (P04486). The sources of the DNAs were as follows: WBP1, p45-11 (11); Cub, pRS306(⌬swp-Cub-dha) (N.J., unpublished data); protein A, p28NZZtrc (19); LexA-VP16, pLexA202ϩVP16 (gift from Alcide Barberis, University of Zürich).…”

Section: Methodsmentioning

confidence: 99%

A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo

Štagljar

Korostensky²,

Johnsson

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

560

462

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Section: Methodsmentioning

confidence: 99%

A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo

Štagljar

Korostensky²,

Johnsson

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

560

462

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“…DNA sequencing was performed as described (13 (14). The shock lysate was passed through an affinity column of human serum albumin (HSA)-Sepharose (9) or of IgG-Sepharose Fast Flow (Pharmacia) as described (15). Flow-through and fractions eluted with 0.5 M HOAc (pH 2.8) were collected and lyophilized.…”

Section: Methodsmentioning

confidence: 99%

“…It is noteworthy that human IGF-I can be efficiently expressed in E. coli without severe proteolysis using the protein A system (15). A comparison of the primary amino acid sequences of the structurally similar IGF-I and -II reveals that the two arginines recognized by a protease in E. coli are present on both molecules.…”

Section: Dual-afminitymentioning

confidence: 99%

Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II.

Hammarberg

Nygren

Holmgren

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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A dual affinity fusion concept has been developed in which the gene encoding the desired product is fused between two flanking heterologous genes encoding IgG-and albumin-binding domains. Using sequential IgG and serum albumin affinity chromatography, a full-length tripartite fusion protein is obtained. This approach was used to recover a full-length fusion product in Escherichia coli containing the human insulin-like growth factor II (IGF-ll). Surprisingly, the recombinant IGF-II showed increased stability against proteolytic degradation in E. coli when produced as a dual affinity fusion protein, as compared to an N-terminal fusion protein.After site-specific cleavage of the tripartite fusion protein, IGF-ll molecules with immunological and receptor binding activity were obtained without renaturation steps. The results demonstrate that proteins can fold into biologically active structures, even if provided with large flanking heterologous protein domains. The concept was further used to characterize the specific degradation of recombinant IGF-II in this heterologous host.

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“…The products are secreted to the periplasm but do not usually pass the outer membrane. Recently, a few gene products have been produced that are also excreted by E. coli (15,19). The genes involved in the excretion originated from Staphylococcus aureus and alkalophilic Bacillus.…”

Section: Introductionmentioning

confidence: 99%

Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli

Biedermann

Jepsen

Riise

et al. 1989

Carlsberg Research Communicati

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The primary structure and physical chemical properties were determined of a nuclease expressed and secreted by Escherichia colt. The plasmid p403-SD2 carried a DNA sequence isolated from Serratia marcescens encoding the enzyme. During cultivation of the E. colt cells, 85% of the enzyme was released to the growth medium. The enzyme was purified and exhibited a single band with a molecular weight about 30,600 daltons on SDS-PAGE similar to nuclease isolated from S. marcescens. The amino acid composition and the amino acid sequence determined directly confirmed the primary structure of 245 amino acids predicted from the DNA sequence, and, in addition, the two disulfide bridges were assigned. Several physical chemical properties were examined. The ability of the enzyme to cross the outer membrane is proposed to depend upon the formation of the proper structures during the folding process.

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Large–Scale Affinity Purification of Human Insulin–Like Growth Factor I from Culture Medium of Escherichia Coli

Cited by 117 publications

References 9 publications

A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo

A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo

Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II.

Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli

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