IGF-1 LR3

What IGF-1 LR3 is

IGF-1 LR3, written in full as Long R3 insulin-like growth factor 1, is a recombinant protein of 83 amino acids derived from human IGF-1, a 70-residue hormone that mediates most of the growth-promoting actions of growth hormone. Two changes define the analogue. A 13-residue extension is added at the N-terminus, which is the "Long" of the name. And the glutamate at position 3 of the native sequence is replaced with arginine, which is the "R3". Both changes were made for a single purpose: to stop the molecule binding to the IGF-binding proteins (IGFBPs) that carry native IGF-1 in serum and in culture medium [1].

The analogue was developed in Adelaide in the late 1980s and early 1990s by the groups of Francis, Ballard and colleagues, originally as a tool for cell culture and as a candidate for enhancing growth and tissue repair in animals [1, 2]. It is produced in Escherichia coli as a recombinant protein, not by chemical synthesis, and it keeps the three disulphide bonds of native IGF-1. Those two facts shape everything about its handling and its certificate, and they distinguish it from the synthetic peptides that make up the rest of the catalogue.

How the literature describes its mechanism

IGF-1 acts through the type 1 IGF receptor, a receptor tyrosine kinase closely related to the insulin receptor. Binding activates the PI3K–Akt and Ras–MAPK pathways, which drive protein synthesis, cell survival, proliferation and differentiation in most tissues. IGF-1 also binds the insulin receptor with lower affinity.

In the body and in serum-containing culture medium, almost all IGF-1 is bound to one of six IGFBPs, which extend its half-life, restrict its access to receptors and modulate its activity in ways that are still being characterised [3]. The IGFBPs bind the N-terminal region of IGF-1, and glutamate 3 is one of the contact residues. Francis and colleagues showed that substituting arginine at that position, and adding the N-terminal extension, reduced IGFBP binding by roughly two orders of magnitude while leaving receptor binding only modestly reduced [1]. The net effect in cell culture is a molecule that is several-fold more potent than IGF-1 itself, not because it binds the receptor better, but because it is not sequestered by the binding proteins in the medium [1]. Tomas and colleagues confirmed the same principle in animals, reporting that LR3 was more anabolic than IGF-1 in catabolic rats at the same dose [2].

For a cell-culture programme the practical consequence is the main reason the analogue is used: in serum-containing or serum-free medium with IGFBPs present, LR3 gives a receptor response that native IGF-1 does not, at a lower concentration, and with less variability between media batches. That property made it a standard supplement in serum-free media for recombinant protein production in CHO cells, which is where most of its routine use lies.

The research record

The LR3 analogue has a defined literature in three areas. The first is the original characterisation of IGF-1 analogues with altered binding-protein affinity, which established the structure–activity relationship described above [1]. The second is the animal work in the early 1990s on growth, nitrogen retention and gut adaptation, in which LR3 was used as a probe for what IGF-1 does when freed from its binding proteins [2]. The third, and by far the largest in practice, is the cell-culture literature, where LR3 is used as a defined replacement for insulin or serum in mammalian cell media and appears in the methods sections of a great many papers on recombinant protein production. There are no clinical trials of IGF-1 LR3; the clinical development of IGF-1 proceeded with the native protein (mecasermin) and with an IGF-1/IGFBP-3 complex.

Regulatory status in the United Kingdom

IGF-1 LR3 has no marketing authorisation and is not a medicine in the UK or anywhere else. Native IGF-1 is a licensed medicine (mecasermin) for a rare growth disorder; the LR3 analogue is not, and BuyUK Peptides does not supply the medicine. The research material is supplied as a chemical for in-vitro laboratory use under the Terms of Sale, not for human or veterinary administration. Purchasers must be 18 or over and are responsible for compliance with the laws and institutional rules that apply to them. Our guide to the UK rules on research compounds sets this out in more detail.

In sport, IGF-1 and its analogues are named explicitly on the WADA Prohibited List under S2, peptide hormones, growth factors and related substances, and are prohibited at all times.

Handling, storage and stability

IGF-1 LR3 is a folded protein with three disulphide bonds, and it needs to be treated as one. The lyophilised powder is supplied in a glass vial with a rubber stopper and an aluminium crimp.

Sealed vial. Store at −20 °C. Short periods at 2–8 °C during shipping and handling do no harm, but a protein of this kind should not be kept at refrigerator temperature for months. Allow the vial to reach room temperature before opening.

Reconstitution. The protein is most soluble in slightly acidic solution. Reconstitute in sterile 10 mM HCl, in 0.1 M acetic acid, or in sterile water, to a stock of 100 µg/mL or more; at neutral pH and low concentration it adsorbs to surfaces and can lose activity. Add the solvent gently and allow the cake to dissolve without agitation; do not vortex a protein. For cell-culture use, dilute the acidic stock into medium containing carrier protein (0.1% BSA or the medium's own protein content) immediately before use. The reconstitution calculator gives volumes.

Reconstituted solution. Aliquot the stock into single-use volumes in low-binding tubes and freeze at −20 °C or −80 °C. Avoid repeated freeze–thaw cycles, which unfold and aggregate the protein; one thaw per aliquot is the rule. A thawed aliquot can be kept at 2–8 °C for a few days.

What to avoid. Bacteriostatic water is not the right solvent for a protein at this scale; the benzyl alcohol it contains can denature folded proteins over time. Do not filter dilute solutions through nylon. Do not expose to reducing agents, which break the disulphide bonds and inactivate the molecule. The storage and stability guide covers proteins separately from synthetic peptides for exactly these reasons.

What a sound certificate for IGF-1 LR3 looks like

Every batch we release carries an independent certificate of analysis, transcribed on the product page and in the certificate register. A certificate for a recombinant protein looks different from one for a synthetic peptide, and some items are specific to it.

Purity. Reversed-phase HPLC is still the usual purity method and should show a single dominant peak at 95% or above; recombinant proteins are typically released at a slightly lower purity specification than short synthetic peptides. SDS-PAGE, reported as a single band at about 9 kDa under non-reducing conditions, is a common supplement. The main impurities to look for are misfolded disulphide isomers, which elute close to the native form, and the dimer.

Identity by LC-MS. The expected mass is about 9,111 Da. Electrospray produces a multiply charged envelope for a protein of this size, and the certificate should report the deconvoluted mass. The observed mass should agree with the theoretical value to within a dalton or so; a mass 6 Da higher than expected indicates reduced disulphides.

Endotoxin. Essential for a recombinant protein made in E. coli, and more important than for a synthetic peptide. The LAL result should be reported in endotoxin units per microgram or milligram, and for cell-culture use a specification of below 1 EU/µg is typical.

Biological activity. Some certificates report a cell-proliferation assay, usually in a line such as FDC-P1 or MCF-7, with an ED50 in the low nanogram-per-millilitre range. This is the one test that confirms the protein is folded and functional; a mass and a purity figure do not.

Residual host-cell protein and DNA. Where reported, these quantify carry-over from the expression host.

Our guide to reading a certificate of analysis goes through a complete report line by line.

Related compounds

IGF-1 LR3 is the final effector in the growth-hormone axis that several catalogue compounds act on upstream. Sermorelin, tesamorelin and CJC-1295 without DAC are growth-hormone-releasing-hormone analogues; ipamorelin and hexarelin are ghrelin receptor agonists. All of them act through the pituitary, and growth hormone in turn induces IGF-1 in the liver; LR3 is used in cell systems to study the IGF-1 receptor directly.

Frequently asked questions

Why is IGF-1 LR3 more potent than IGF-1 in cell culture? Not because it binds the receptor better, but because it does not bind the IGF-binding proteins in the medium, so more of it is free to reach the receptor.

Is it a peptide or a protein? At 83 residues with three disulphide bonds it is a small protein, made recombinantly rather than synthesised, and it should be handled as a protein.

Can I reconstitute it in bacteriostatic water? It is better not to. Use dilute acid or sterile water, aliquot, and freeze.

Why does the certificate quote a cell-based activity? Because mass and purity do not show that the protein is correctly folded. A proliferation assay does.

References

1. Francis GL, Ross M, Ballard FJ, et al. 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. Journal of Molecular Endocrinology. 1992;8(3):213–223. doi:10.1677/jme.0.0080213.
2. Tomas FM, Knowles SE, Owens PC, et al. Insulin-like growth factor-I (IGF-I) and especially IGF-I variants are anabolic in dexamethasone-treated rats. Biochemical Journal. 1992;282(1):91–97. doi:10.1042/bj2820091.
3. Clemmons DR. Role of IGF-binding proteins in regulating IGF responses to changes in metabolism. Journal of Molecular Endocrinology. 2018;61(1):T139–T169. doi:10.1530/JME-18-0016.
4. Laron Z. Insulin-like growth factor 1 (IGF-1): a growth hormone. Molecular Pathology. 2001;54(5):311–316. doi:10.1136/mp.54.5.311.

This monograph summarises the published literature for laboratory researchers. It is not medical advice, and nothing on this site is for human or veterinary use.