IGF-1 LR3 1mg

What is IGF-1 LR3 ?

IGF-1 LR3 (Long R3 IGF-1) is an 83 amino acid analogue of human insulin-like growth factor 1, supplied as a 1mg lyophilised powder. An Arg3 substitution and a 13 residue N-terminal extension cut its binding to IGF binding proteins about 1000-fold. That makes it a standard reagent for IGF-1 receptor research and serum-free cell culture.

The literature writes it as Long-[Arg3]-IGF-I or LR3IGF-I. It is native 70 residue IGF-I with two changes: arginine replaces glutamate at position 3, and a 13 residue extension, MFPAMPLLSLFVN, sits on the N-terminus. The extension is the first 11 residues of methionyl porcine growth hormone plus Val-Asn. It is left over from the fusion used to express the protein in Escherichia coli (Francis et al., J Mol Endocrinol, 1992). Both changes sit in the region that IGF binding proteins contact.

IGF-1 LR3 Research Applications

• Serum-free culture : LongR3 sustained the viability of two recombinant CHO cell lines better than insulin under production conditions (Morris and Schmid, 2000).
• Receptor signalling : a reagent for type 1 IGF receptor activation and downstream PI3K and MAPK signalling.
• Binding protein biology : separating IGFBP sequestration from receptor pharmacology, the comparison the analogue was built to make.

Published in-vivo work is confined to animal models. There are no human clinical trials of IGF-1 LR3.

IGF-1 LR3 vs Native IGF-1 and the IGF Binding Protein Problem

Native IGF-I does not circulate free. Most of it is held by IGF binding proteins, mainly IGFBP-3, which limit its access to the receptor. In any system containing serum or secreted IGFBPs, the concentration you add is not the concentration the receptor sees. Ballard et al. (Growth Regulation, 1993) found IGF-I binds IGFBP-3 with about 1000-fold higher affinity than LR3IGF-I. In L6 myoblasts, that gave LR3IGF-I 5 to 10-fold greater potency.

The gain depends on the binding proteins. In chicken embryo fibroblasts, which secrete no detectable IGFBPs, Long [Arg3]-IGF-I was less potent than IGF-I (Francis et al., 1992). The trade-off is that IGF-1 LR3 is a poor model of native IGF-I physiology. Use it for receptor pharmacology and serum-free culture, and native IGF-I to study the binding protein axis itself.

Research Overview

IGF-1 LR3, written in the primary literature as Long-[Arg3]-IGF-I or LR3IGF-I, is an 83 residue recombinant analogue of human insulin-like growth factor 1. It differs from the native 70 residue growth factor in two respects: glutamate at position 3 is replaced by arginine, and a hydrophobic 13 residue extension (MFPAMPLLSLFVN) is fused to the N-terminus. The analogue was characterised by Francis and colleagues in the Journal of Molecular Endocrinology in 1992. The extension is not a rationally designed pharmacophore, it is a residual fragment of the methionyl porcine growth hormone fusion partner used to express the protein in Escherichia coli, characterised in a companion paper in the same journal and year. Both modifications sit in the region that IGF binding proteins recognise, and the functional consequence is a large loss of IGFBP affinity while type 1 IGF receptor binding is largely preserved. Ballard and colleagues (Growth Regulation, 1993) reported that native IGF-I binds IGFBP-3 with approximately one thousand times the affinity of LR3IGF-I. Since the great majority of circulating IGF-I is normally sequestered in IGFBP complexes, an analogue that escapes that buffering behaves very differently in any system containing binding proteins, and that is the reason the IGF 1 LR3 variant exists as a reagent at all. The three disulfide bridges of the IGF-I core are retained, which is why the molecular formula carries nine sulfur atoms, six from cysteine and three from methionine. The best established application of this IGF 1 peptide is industrial rather than physiological: LONG R3 IGF-I is a standard growth factor supplement in serum-free CHO and HEK293 culture, where Morris and Schmid (Biotechnology Progress, 2000) compared it directly against insulin. Published in-vivo work is confined to rodent, pig and non-human primate models. There are no published human clinical trials of IGF-1 LR3, and it holds no marketing authorisation in any jurisdiction.

Storage and Reconstitution

Store the sealed 1mg vial at -20°C. Three methionines, two of them in the N-terminal extension, make dilute solutions prone to oxidation, so minimise headspace air and keep solutions cold and dark. Freeze-thaw cycles promote aggregation in disulfide-bonded proteins more than in short peptides, so freeze single-use aliquots.

Vials are sold on their own, without bacteriostatic water for mixing or other supplies.

How to reconstitute and handle

IGF-1 LR3 is a folded 9.1 kDa protein with three disulfide bridges, so agitation is the main hazard. Let the vial reach room temperature, run bacteriostatic water slowly down the inside wall and swirl gently until clear. Never shake: shear and air-liquid interface exposure drive aggregation. Dilute working solutions lose material to untreated plastic and glass, so use low-binding labware and prepare dilutions just before use.

• Allow the vial to reach room temperature before opening so condensation does not settle on the cake.
• Treat this as a folded protein, not a short synthetic peptide. At roughly 9.1 kDa with three disulfide bridges, IGF-1 LR3 is vulnerable to shear and to air-liquid interface denaturation, which is why agitation is the single most common cause of a cloudy or aggregated preparation.
• Expect surface adsorption losses at low concentration. Dilute working solutions of an 83 residue growth factor lose measurable material to untreated polypropylene and glass, so use low-binding labware and prepare dilutions immediately before use rather than storing them.
• The sequence carries three methionine residues, two of them in the N-terminal extension, all oxidation-sensitive. Minimise headspace air, protect from light, and do not leave dilute solutions standing at room temperature.
• Store the reconstituted solution at 2-8°C and prepare single-use aliquots at -20°C for longer holding. Avoid repeated freeze-thaw cycles, which promote aggregation in disulfide-bonded proteins.
• Handle under aseptic conditions, swabbing the stopper of both vials with alcohol before each needle entry. The site reconstitution guide and reconstitution calculator cover the full aseptic workflow and dilution arithmetic.

For the full method, see the peptide reconstitution guide and the reconstitution calculator.

Research References

• 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 (J Mol Endocrinol, 1992)
• Effects of interactions between IGFBPs and IGFs on the plasma clearance and in vivo biological activities of IGFs and IGF analogs (Growth Regul, 1993)
• Effects of insulin and LongR3 on serum-free Chinese hamster ovary cell cultures expressing two recombinant proteins (Biotechnol Prog, 2000)

Published research on IGF-1 LR3 is indexed on PubMed. See our full UK buyer's guide for IGF-1 LR3 and UK buyer's guide for IGF-1 LR3 CoA.