scholarly journals Enhanced proliferative effects of a baculovirus-produced fusion protein of insulin-like growth factor and α1-proteinase inhibitor and improved anti-elastase activity of the inhibitor with glutamate at position 351

2002 ◽  
Vol 15 (5) ◽  
pp. 413-418 ◽  
Author(s):  
C. Sandoval ◽  
H. Curtis ◽  
L.F. Congote
Keyword(s):  
Growth Factor ◽  
Fusion Protein ◽  
Proteinase Inhibitor ◽  
Insulin Like Growth Factor ◽  
Elastase Activity

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 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.

Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency

1992 ◽  
Vol 8 (3) ◽  
pp. 213-223 ◽  
Author(s):  
G. L. Francis ◽  
M. Ross ◽  
F. J. Ballard ◽  
S. J. Milner ◽  
C. Senn ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fusion Protein ◽  
Fusion Peptide ◽  
Biologically Active ◽  
Peptide Sequence ◽  
Insulin Like Growth Factor ◽  
Fusion Peptides ◽  
Igf I ◽  
Igf Binding ◽  
Biological Potency

ABSTRACT An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1–11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1–11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1–11)-Val-Asn-[Arg3]-IGF-I, where Glu3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1–3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]IGF-I-des(1–3)IGF-I>Long [Gly3]-IGF-I>Long IGF-I>IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.

scholarly journals Chemical heterogeneity as a result of hydroxylamine cleavage of a fusion protein of human insulin-like growth factor I

1992 ◽  
Vol 285 (1) ◽  
pp. 207-213 ◽  
Author(s):  
E Canova-Davis ◽  
M Eng ◽  
V Mukku ◽  
D H Reifsnyder ◽  
C V Olson ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fusion Protein ◽  
Human Insulin ◽  
Recombinant Dna ◽  
Fermentation Broth ◽  
Reversed Phase ◽  
Staphylococcal Protein A ◽  
Insulin Like Growth Factor ◽  
Factor I ◽  
Growth Factor I

Recombinant DNA techniques were used to biosynthesize human insulin-like growth factor I (hIGF-I) as a fusion protein wherein the fusion polypeptide is an IgG-binding moiety derived from staphylococcal protein A. This fusion protein is produced in Escherichia coli and secreted into the fermentation broth. In order to release mature recombinant-derived hIGF-I (rhIGF-I), the fusion protein is treated with hydroxylamine, which cleaves a susceptible Asn-Gly bond that has been engineered into the fusion protein gene. Reversed-phase h.p.l.c. was used to estimate the purity of the rhIGF-I preparations, especially for the quantification of the methionine sulphoxide-containing variant. It was determined that hydroxylamine cleavage of the fusion protein produced, as a side reaction, hydroxamates of the asparagine and glutamine residues in rhIGF-I. Although isoelectric focusing was effective in detecting, and reversed-phase h.p.l.c. for producing enriched fractions of the hydroxamate variants, ion-exchange chromatography was a more definitive procedure, as it allowed quantification and facile removal of these variants. The identity of the variants as hydroxamates was established by Staphylococcus aureus V8 proteinase digestion, followed by m.s., as the modification was transparent to amino acid and N-terminal sequence analyses. The biological activity of rhIGF-I was established by its ability to incorporate [3H]thymidine into the DNA of BALB/c373 cells and by a radioreceptor assay utilizing human placental membranes. Both assays demonstrate that the native, recombinant and methionine sulphoxide and hydroxamate IGF-I variants are essentially equipotent.

scholarly journals Expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4

2001 ◽  
Vol 168 (2) ◽  
pp. 283-296 ◽  
Author(s):  
D Chelius ◽  
MA Baldwin ◽  
X Lu ◽  
EM Spencer
Keyword(s):  
Mass Spectrometry ◽  
Growth Factor ◽  
Recombinant Protein ◽  
Fusion Protein ◽  
Affinity Chromatography ◽  
Cysteine Residues ◽  
Insulin Like Growth Factor ◽  
Disulfide Linkages ◽  
N Terminus ◽  
Igf I

Insulin-like growth factor binding protein-4 (IGFBP-4), like the other five IGFBPs, is a critical regulator of the activity of insulin-like growth factor (IGF)-I and IGF-II. However IGFBP-4 seems to be the only IGFBP with no potential to enhance the mitogenic actions of the IGFs. IGFBP-1 to -3 and -5 each contain 18 conserved cysteine residues, IGFBP-6 lacks two of the twelve N-terminal cysteines, while IGFBP-4 has two additional cysteines in the central region. A plasmid was constructed to express rat IGFBP-4 as a thioredoxin fusion protein that included a hexahistidine sequence to permit affinity purification. The fusion protein was expressed in E.coli, purified using nickel-chelate affinity chromatography and cleaved by tobacco etch virus (TEV) protease to produce mature rat IGFBP-4 with an additional glycine residue at the N-terminus. Final purification was achieved by further nickel affinity chromatography and reverse phase HPLC. The isoelectric points of the recombinant IGFBP-4 were the same as those of the non-glycosylated isoforms of IGFBP-4 in rat serum. The binding affinities of the recombinant protein and IGFBP-4 secreted by rat cells to IGF-I were compared using a newly developed binding assay. No significant difference could be detected, consistent with proper folding of the recombinant protein. This indicates that glycosylation of IGFBP-4 does not affect its binding to IGF-I. Using mass spectrometry and tandem mass spectrometry no differences between authentic and recombinant IGFBP-4 could be detected. Eight of the ten disulfide linkages have been determined, including linkages of conserved cysteine residues not previously identified in other IGFBPs. Numbering the cysteine residues sequentially from the N-terminus only the disulfide connectivity of C1, C2, C5 and C6 could not be determined. However, C1 is not linked to C1 and C5 is not linked to C6. The established linkages were C3 to C8, C4 to C7, C9 to C 11, and C10 to C12. The two cysteines in the non-conserved mid-region unique to IGFBP-4 (C13 and C14) are linked together. Linkage of the C-terminal cysteine residues is identical to that of IGFBP-2, -5 and -6 (C15 to C16, C17 to C18 and C19 to C20). The central flexible core of IGFBP-4, containing two additional cysteines may contribute to its unique biological action.

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