scholarly journals Enhanced Production of Insulin-Like Growth Factor I Fusion Protein in Escherichia coli by Coexpression of the Down-Regulated Genes Identified by Transcriptome Profiling

2003 ◽  
Vol 69 (8) ◽  
pp. 4737-4742 ◽  
Author(s):  
Jong Hyun Choi ◽  
Sang Jun Lee ◽  
Seok Jae Lee ◽  
Sang Yup Lee
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Fusion Protein ◽  
Transcriptome Profiling ◽  
Insulin Like Growth Factor ◽  
Gene Encoding ◽  
Factor I ◽  
Enhanced Production ◽  
Igf I ◽  
Growth Factor I

ABSTRACT The transcriptome profiles of recombinant Escherichia coli producing human insulin-like growth factor I fusion protein (IGF-If) during the high-cell-density fed-batch culture were analyzed using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly down-regulated during the production of IGF-If after induction. Among these down-regulated genes, we rationally selected and coexpressed in E. coli producing IGF-If the prsA gene (encoding a phosphoribosyl pyrophosphate synthetase) and the glpF gene (encoding a glycerol transporter), which are involved in an early key step in the biosynthetic pathway of nucleotides and amino acids (Trp and His) and the first step in glycerol utilization, respectively. As a result, the production of IGF-If could be increased from 1.8 ± 0.13 (± standard deviation) to 4.3 ± 0.24 g/liter. The volumetric productivity was also increased from 0.36 ± 0.027 to 0.82 ± 0.048 g/liter/h. These results demonstrate that transcriptome profiling can provide invaluable information in designing engineered strains showing enhanced performance.

Production and characterization of recombinant insulin-like growth factor-I (IGF-I) and potent analogues of IGF-I, with Gly or Arg substituted for Glu 3, following their expression in Escherichia coli as fusion proteins

1992 ◽  
Vol 8 (1) ◽  
pp. 29-41 ◽  
Author(s):  
R. King ◽  
J. R. E. Wells ◽  
P. Krieg ◽  
M. Snoswell ◽  
J. Brazier ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Expression System ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Igf I ◽  
Igf Binding ◽  
Growth Factor I

ABSTRACT The development of an efficient expression system for insulin-like growth factor-I (IGF-I) in Escherichia coli as a fusion protein is described. The fusion protein consists of an N-terminal extension made up of the first 46 amino acids of methionyl porcine GH ([Met1]-pGH) followed by the dipeptide Val-Asn. The latter two residues provide a unique hydroxylamine-sensitive link between [Met1]-pGH(1-46) and the N-terminal Gly of IGF-I. Downstream processing of the fusion proteins involved isolation of inclusion bodies, cleavage at the Asn-Gly bond, refolding of the reduced IGF-I peptide and purification to homogeneity. This expression system was also used to produce two variants of IGF-I in which Glu3 was substituted by either Gly or Arg to give [Gly3]-IGF-I and [Arg3]-IGF-I respectively. Production of milligram quantities of IGF-I peptide was readily achieved. The purity of the IGF-I, [Gly3]-IGF-I and [Arg3]-IGF-I was established by high-performance liquid chromatography and N-terminal sequence analysis. [Gly3]-IGF-I and [Arg3]-IGF-I were more potent than IGF-I in biological assays measuring stimulation of protein synthesis and DNA synthesis or inhibition of protein breakdown in rat L6 myoblasts. Both analogues bound very poorly to bovine IGF-binding protein-2 and slightly less well than IGF-I to the type-1 receptor on rat L6 myoblasts. We conclude that reduced binding to IGF-binding proteins rather than increased receptor binding is the likely explanation for the greater biological potency of the analogues compared with IGF-I.

scholarly journals Separation and characterization of modified variants of recombinant human insulin-like growth factor I derived from a fusion protein secreted from Escherichia coli

1990 ◽  
Vol 271 (2) ◽  
pp. 357-363 ◽  
Author(s):  
G Forsberg ◽  
G Palm ◽  
A Ekebacke ◽  
S Josephson ◽  
M Hartmanis
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Fusion Protein ◽  
Human Insulin ◽  
Insulin Like Growth Factor ◽  
Recombinant Human Insulin ◽  
Factor I ◽  
Methionine Residue ◽  
Igf I ◽  
Growth Factor I

Human insulin-like growth factor I, IGF-I, was produced in Escherichia coli fused to a synthetic IgG-binding peptide The fusion protein is secreted into the medium during fermentation and was initially purified on an IgG-Sepharose column. After hydroxylamine cleavage, IGF-I was purified to homogeneity. During purification, impurities in the form of modified variants of IGF-I were detected and characterized. The closely related impurities were identified to be a misfolded form of IGF-I, having mismatched disulphide bonds, a form with the single methionine residue in IGF-I oxidized to methionine sulphoxide and a variant in which the methionine residue was substituted by a norleucine residue during protein synthesis. A form proteolytically cleaved between two arginine residue was also detected. These impurities were separated from the major component, native IGF-I, by using reverse-phase h.p.l.c. The modified molecules as well as native IGF-I were characterized both as intact molecules and as fragments, after pepsin digestion, using the techniques of plasma desorption m.s., N-terminal sequencing and amino acid analysis. The oxidized form was 90%, and the norleucine analogue was 70%, as potent as native IGF-I in a biological radioreceptor assay, and the form having mismatched disulphides lacked receptor affinity.

Recombinant gilthead seabream (Sparus aurata) insulin-like growth factor-I: subcloning, expression in Escherichia coli, purification and characterization

1997 ◽  
Vol 153 (1) ◽  
pp. 139-150 ◽  
Author(s):  
M Fine ◽  
R Amuly ◽  
Y Sandowski ◽  
T A Marchant ◽  
S J Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Factor ◽  
Sparus Aurata ◽  
Biologically Active ◽  
Gilthead Seabream ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Prokaryotic Expression Vector ◽  
Igf I ◽  
Growth Factor I

Abstract Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding for the mature protein was cloned in a pGEM-3Z vector, and then transferred into prokaryotic expression vector pET-11a and expressed in Escherichia coli BL21(DE3) cells upon induction with isopropyl thiogalactoside. The expressed protein contained within the inclusion-body pellet was solubilized in 4·5 m urea, refolded for 24 h at pH 11·3 in the presence of catalytic amounts of cysteine and purified to over 98% purity, as a monomeric methionyl-gsIGF-I. Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein. Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins. In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was ∼200-fold lower than that of hIGF-I. Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells. In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active. Journal of Endocrinology (1997) 153, 139–150

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