GHK-Cu as a multifunctional copper peptide: synthesis routes, process engineering and emerging applications

Abstract

Glycyl-L-histidyl-L-lysine–copper (GHK-Cu), an endogenous tripeptide–metal complex, has attracted increasing interest due to its multifaceted biological roles in tissue repair, anti-inflammatory regulation, extracellular matrix remodeling, and redox homeostasis. In recent years, GHK-Cu has evolved from a dermatological bioactive ingredient into a representative model system linking coordination chemistry, peptide bioactivity, and biomanufacturing engineering. This review provides a comprehensive synthesis of the molecular mechanisms, production strategies, quality-control frameworks, and emerging applications of GHK-Cu in advanced biomaterials and delivery systems. Particular emphasis is placed on the key technologies enabling the transition from chemical synthesis to scalable biomanufacturing, including tandem-repeat expression, high-cell-density fermentation, inclusion-body-assisted purification, and Cu(II) complex stabilization. Analytical approaches for structural verification and impurity profiling, as well as global regulatory classifications and formulation-stability considerations, are also summarized. Remaining challenges in clinical translation, production standardization, and large-scale quality assurance are highlighted. This work aims to provide a systematic and forward-looking framework to support the industrial and biomedical deployment of copper–peptide therapeutics.

Graphical abstract

Keywords

GHK-Cu / Copper peptide / Biomanufacturing / Tandem repeat expression / Regenerative biomaterials

References

[1] Alshammari N, Platts JA. Theoretical study of copper binding to GHK peptide. Comput Biol Chem. 2020, 86. 107265

[2] Das A. GHK-Cu (Copper Peptide) market by purity level, product type and application: global market size, share, growth, trends and statistical analysis report by region and segment forecasts 2025–2033. Cognitive Market Research, 2025. https://www.cognitivemarketresearch.com/copper-peptide-ghk-cu-market-report

[3] Baeshen NA, Baeshen MN, Sheikh A, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Redwan EM. Cell factories for insulin production. Microb Cell Fact. 2014, 13(1. 141

[4] Baeshen MN, Al-Hejin AM, Bora RS, Ahmed MM, Ramadan HA, Saini KS, Baeshen NA, Redwan EM. Production of biopharmaceuticals in E. coli: current scenario and future perspectives. J Microbiol Biotechnol. 2015, 257): 953-962.

[5] Ben David A, Papir Y, Hazan O, Redelman M, Diamant E, Barnea A, Torgeman A, Zichel R. High cell density cultivation process for the expression of botulinum neurotoxin a receptor binding domain. Toxins. 2022, 14(4. 281

[6] Bhatwa A, Wang W, Hassan YI, Abraham N, Li XZ, Zhou T. Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications. Front Bioeng Biotechnol. 2021, 9. 630551

[7] Bossak-Ahmad K, Wisniewska MD, Bal W, Drew SC, Fraczyk T. Ternary Cu(II) complex with GHK peptide and cis-urocanic acid as a potential physiologically functional copper chelate. Int J Mol Sci. 2020, 2117. 6190

[8] Boyd SD, Ullrich MS, Skopp A, Winkler DD. Copper sources for Sod1 activation. Antioxidants. 2020, 96. 500

[9] C.A.o.S. Institute of Biophysics, High-purity preparation method of recombinant blue copper peptide precursor-oligopeptide (GHK), China Patent, Beijing, China. 2021.

[10] Cao HY, Lian JN, Lin ZS, Zhu ZW, Wang H,Wang HY. A method for high-density fermentation production of semaglutide precursor, China Patent. 2021.

[11] Cao HY, Lian JN, Lin ZS, Zhu ZW, Wang H, Zhang SY. A fusion protein and its application in the preparation of semaglutide precursor, China. 2021.

[12] Carlson ED, Gan R, Hodgman CE, Jewett MC. Cell-free protein synthesis: Applications come of age. Biotechnol Adv. 2012, 305): 1185-1194.

[13] Chen H, Yang P, Xue P, Li S, Dan X, Li Y, Lei L, Fan X. Food-derived tripeptide-copper self-healing hydrogel for infected wound healing. Biomater Res. 2025, 29. 0139

[14] Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001, 30(3): 665-676.

[15] Clough R, Harrington CF, Hill SJ, Madrid Y, Tyson JF. Atomic spectrometry update: review of advances in elemental speciation. J Anal at Spectrom. 2021, 36(7): 1326-1373.

[16] Conlon JM. Purification of naturally occurring peptides by reversed-phase HPLC. Nat Protoc. 2007, 2(1): 191-197.

[17] de Castro Bras LE, Frangogiannis NG. Extracellular matrix-derived peptides in tissue remodeling and fibrosis. Matrix Biol. 2020, 91: 176-187.

[18] De Feo CJ, Aller SG, Siluvai GS, Blackburn NJ, Unger VM. Three-dimensional structure of the human copper transporter hCTR1. Proc Natl Acad Sci U S A. 2009, 106(11): 4237-4242.

[19] De Luca C, Lievore G, Bozza D, Buratti A, Cavazzini A, Ricci A, Macis M, Cabri W, Felletti S, Catani M. Downstream processing of therapeutic peptides by means of preparative liquid chromatography. Molecules. 2021, 26(15. 4688

[20] Dou Y, Lee A, Zhu L, Morton J, Ladiges W. The potential of GHK as an anti-aging peptide. Aging Pathobiol Ther. 2020, 21): 58-61.

[21] Dymek M, Olechowska K, Hac-Wydro K, Sikora E. Liposomes as carriers of GHK-Cu tripeptide for cosmetic application. Pharmaceutics. 2023, 15(10. 2485

[22] Faller P, Hureau C. Bioinorganic chemistry of copper and zinc ions coordinated to amyloid-beta peptide. Dalton Trans. 2009, 77): 1080-1094.

[23] Fenton HJH. LXXIII: Oxidation of tartaric acid in presence of iron. J Chem Soc Trans. 1894, 65(0): 899-910.

[24] Festa RA, Thiele DJ. Copper: an essential metal in biology. Curr Biol. 2011, 21(21): R877-R883.

[25] Frantz C, Stewart KM, Weaver VM. The extracellular matrix at a glance. J Cell Sci. 2010, 123(24): 4195-4200.

[26] Gaetke LM, Chow CK. Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology. 2003, 189(1-2): 147-163.

[27] Gopal GJ, Kumar A. Strategies for the production of recombinant protein in Escherichia coli. Protein J. 2013, 32(6): 419-425.

[28] Harford C, Sarkar B. Amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif of proteins and peptides: metal binding, DNA cleavage, and other properties. Acc Chem Res. 1997, 30(3): 123-130.

[29] He L, Feng A, Guo H, Huang H, Deng Q, Zhao E, Yang M. LRG1 mediated by ATF3 promotes growth and angiogenesis of gastric cancer by regulating the SRC/STAT3/VEGFA pathway. Gastric Cancer. 2022, 25(3): 527-541.

[30] Hostynek JJ, Dreher F, Maibach HI. Human skin penetration of a copper tripeptide in vitro as a function of skin layer. Inflamm Res. 2011, 60(1): 79-86.

[31] Hsiao CD, Wu HH, Malhotra N, Liu YC, Wu YH, Lin YN, Saputra F, Santoso F, Chen KH. Expression and purification of recombinant GHK tripeptides are able to protect against acute cardiotoxicity from exposure to waterborne-copper in zebrafish. Biomolecules. 2020, 10(9. 1202

[32] Hwang PM, Pan JS, Sykes BD. Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli. FEBS Lett. 2014, 5882): 247-252.

[33] Irving H, Williams RJP. 637. The stability of transition-metal complexes. J Chem Soc. 1953.

[34] Isidro-Llobet A, Kenworthy MN, Mukherjee S, Kopach ME, Wegner K, Gallou F, Smith AG, Roschangar F. Sustainability challenges in peptide synthesis and purification: From R&D to production. J Org Chem. 2019, 84(8): 4615-4628.

[35] Islam R, Bilal H, Wang X, Zhang L. Tripeptides Ghk and GhkCu-modified silver nanoparticles for enhanced antibacterial and wound healing activities. Colloids Surf B Biointerfaces. 2024, 236. 113785

[36] Jiang F, Wu Y, Liu Z, Hong M, Huang Y. Synergy of GHK-Cu and hyaluronic acid on collagen IV upregulation via fibroblast and ex-vivo skin tests. J Cosmet Dermatol. 2023, 22(9): 2598-2604.

[37] Jing J, Chen Y, Sheng L, Wu M. Optimized production of insulin variant, a recombinant platelet aggregation inhibitor, by high cell-density fermentation of recombinant Escherichia coli. Protein Expr Purif. 2018, 152: 7-12.

[38] Kachhawaha K, Singh S, Joshi K, Nain P, Singh SK. Bioprocessing of recombinant proteins from Escherichia coli inclusion bodies: Insights from structure-function relationship for novel applications. Prep Biochem Biotechnol. 2023, 53(7): 728-752.

[39] Kehrer JP. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology. 2000, 149(1): 43-50.

[40] Kekessie I, Wegner K, Martinez I, Kopach ME, White TD, Tom JK, Kenworthy MN, Gallou F, Lopez J, Koenig SG, Payne PR, Eissler S, Arumugam B, Li C, Mukherjee S, Isidro-Llobet A, Ludemann-Hombourger O, Richardson P, Kittelmann J, Sejer Pedersen D, van den Bos LJ. Process mass intensity (PMI): a holistic analysis of current peptide manufacturing processes informs sustainability in peptide synthesis. J Org Chem. 2024, 89(7): 4261-4282.

[41] Kopp J, Kolkmann AM, Veleenturf PG, Spadiut O, Herwig C, Slouka C. Boosting recombinant inclusion body production-from classical fed-batch approach to continuous cultivation. Front Bioeng Biotechnol. 2019, 7. 297

[42] Kremennaya MA, Soldatov MA, Podkovyrina YS, Dadasheva IA, Soldatov AV. X-ray spectroscopy study of the Cu(II)GHK peptide complex. J Struct Chem. 2017, 58(6): 1213-1219.

[43] L. Shanghai Yieryi Biotechnology Co., A method for synthesizing GHK tripeptide, Shanghai Yieryi Biotechnology Co., Ltd., China. 2014.

[44] L. Nanjing GenScript Biotechnology Co., A method for synthesizing GHK-Cu, Nanjing GenScript Biotechnology Co., Ltd., China. 2022.

[45] L. Wuxi Leshi Biotechnology Co., A GHK-Cu and its synthesis method, Wuxi Leshi Biotechnology Co., Ltd., China. 2023.

[46] Lau SJ, Sarkar B. The interaction of copper(II) and glycyl-L-histidyl-L-lysine, a growth-modulating tripeptide from plasma. Biochem J. 1981, 199(3): 649-656.

[47] Lee SY. High cell-density culture of Escherichia coli. Trends Biotechnol. 1996, 14(3): 98-105.

[48] Lee S, Lee SM, Lee SH, Choi WK, Park SJ, Kim DY, Oh SW, Oh J, Cho JY, Lee J, Chien PN, Nam SY, Heo CY, Lee YS, Kwak EA, Chung WJ. In situ photo-crosslinkable hyaluronic acid-based hydrogel embedded with GHK peptide nanofibers for bioactive wound healing. Acta Biomater. 2023, 172: 159-174.

[49] Liu Y, Ren L, Ge L, Cui Q, Cao X, Hou Y, Bai F, Bai G. A strategy for fusion expression and preparation of functional glucagon-like peptide-1 (GLP-1) analogue by introducing an enterokinase cleavage site. Biotechnol Lett. 2014, 36(8): 1675-1680.

[50] Liu T, Hu L, Lu B, Bo Y, Liao Y, Zhan J, Pei Y, Sun H, Wang Z, Guo C, Zhang J. A novel delivery vehicle for copper peptides. New J Chem. 2023, 47(1): 75-83.

[51] Lu P, Takai K, Weaver VM, Werb Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb Perspect Biol. 2011, 3(12. a005058

[52] Lutsenko S. Human copper homeostasis: A network of interconnected pathways. Curr Opin Chem Biol. 2010, 142): 211-217.

[53] Ma WH, Li M, Ma HF, Li W, Liu L, Yin Y, Zhou XM, Hou G. Protective effects of GHK-Cu in bleomycin-induced pulmonary fibrosis via anti-oxidative stress and anti-inflammation pathways. Life Sci. 2020, 241. 117139

[54] Mant CT, Chen Y, Yan Z, Popa TV, Kovacs JM, Mills JB, Hodges RS. HPLC analysis and purification of peptides. Peptide characterization and application protocols. 2007, Totowa, NJ, Humana Press355

[55] Maquart FX, Pickart L, Laurent M, Gillery P, Monboisse JC, Borel JP. Stimulation of collagen synthesis in fibroblast cultures by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+. FEBS Lett. 1988, 238(2): 343-346.

[56] Meyerstein D. What are the oxidizing intermediates in the Fenton and Fenton-like reactions? A perspective. Antioxidants. 2022, 11(7. 1368

[57] Min JH, Sarlus H, Harris RA. Glycyl-l-histidyl-l-lysine prevents copper- and zinc-induced protein aggregation and central nervous system cell death in vitro. Metallomics. 2024, 165. mfae019

[58] Panda AK. Bioprocessing of therapeutic proteins from the inclusion bodies of Escherichia coli. Adv Biochem Eng Biotechnol. 2003, 85: 43-93.

[59] Pickart L, Margolina A. Skin regenerative and anti-cancer actions of copper peptides. Cosmetics. 2018, 52): 29.

[60] Pickart L, Thaler M. Tripeptide in human serum which prolongs survival of normal liver cells and stimulates growth in neoplastic liver. Nat New Biol. 1973, 24312485-87

[61] Pickart L, Thayer L, Thaler MM. A synthetic tripeptide which increases survival of normal liver cells, and stimulates growth in hepatoma cells. Biochem Biophys Res Commun. 1973, 54(2): 562-566.

[62] Pickart L, Vasquez-Soltero JM, Margolina A. The human tripeptide GHK-Cu in prevention of oxidative stress and degenerative conditions of aging: implications for cognitive health. Oxid Med Cell Longev. 2012, 2012(1. 324832

[63] Pickart L, Vasquez-Soltero JM, Margolina A. GHK peptide as a natural modulator of multiple cellular pathways in skin regeneration. Biomed Res Int. 2015, 20151. 648108

[64] Pickart LR. Method of using copper (II) containing compounds to accelerate wound healing. Google Patents. 1992.

[65] Pollard JD, Quan S, Kang T, Koch RJ. Effects of copper tripeptide on the growth and expression of growth factors by normal and irradiated fibroblasts. Arch Facial Plast Surg. 2005, 71): 27-31.

[66] Raducanu VS, Raducanu DV, Ouyang Y, Tehseen M, Takahashi M, Hamdan SM. TSGIT: an N- and C-terminal tandem tag system for purification of native and intein-mediated ligation-ready proteins. Protein Sci. 2021, 30(2): 497-512.

[67] Riesenberg D. High-cell-density cultivation of Escherichia coli. Curr Opin Biotechnol. 1991, 2(3): 380-384.

[68] Riesenberg D, Schulz V, Knorre WA, Pohl HD, Korz D, Sanders EA, Ross A, Deckwer WD. High cell density cultivation of Escherichia coli at controlled specific growth rate. J Biotechnol. 1991, 20(1): 17-27.

[69] Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014, 5. 172

[70] Saraswat M, Musante L, Ravida A, Shortt B, Byrne B, Holthofer H. Preparative purification of recombinant proteins: current status and future trends. Biomed Res Int. 2013, 2013(1. 312709

[71] Schneier M, Razdan S, Miller AM, Briceno ME, Barua S. Current technologies to endotoxin detection and removal for biopharmaceutical purification. Biotechnol Bioeng. 2020, 1178): 2588-2609.

[72] Shiloach J, Fass R. Growing E. coli to high cell density: A historical perspective on method development. Biotechnol Adv. 2005, 23(5): 345-357.

[73] Shiloach J, Kaufman J, Guillard AS, Fass R. Effect of glucose supply strategy on acetate accumulation, growth, and recombinant protein production by Escherichia coli BL21 (lambdaDE3) and Escherichia coli JM109. Biotechnol Bioeng. 1996, 49(4): 421-428.

[74] Siew YY, Zhang W. Downstream processing of recombinant human insulin and its analogues production from E. coli inclusion bodies. Bioresour Bioprocess. 2021, 8(1. 65

[75] Silverman AD, Karim AS, Jewett MC. Cell-free gene expression: An expanded repertoire of applications. Nat Rev Genet. 2020, 213): 151-170.

[76] Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microbial Cell Fact. 2015, 14(1. 41

[77] Slouka C, Kopp J, Spadiut O, Herwig C. Perspectives of inclusion bodies for bio-based products: curse or blessing?. Appl Microbiol Biotechnol. 2019, 1033): 1143-1153.

[78] Son YJ, Phue JN, Trinh LB, Lee SJ, Shiloach J. The role of Cra in regulating acetate excretion and osmotic tolerance in E. coli K-12 and E. coli B at high density growth. Microb Cell Fact. 2011, 10(1. 52

[79] Springfield E, Willis A, Merle J, Mazlo J, Ngu-Schwemlein M. Spectroscopic and theoretical studies of Hg(II) complexation with some dicysteinyl tetrapeptides. Bioinorg Chem Appl. 2021, 20211. 9911474

[80] Tsvetkov P, Coy S, Petrova B, Dreishpoon M, Verma A, Abdusamad M, Rossen J, Joesch-Cohen L, Humeidi R, Spangler RD, Eaton JK, Frenkel E, Kocak M, Corsello SM, Lutsenko S, Kanarek N, Santagata S, Golub TR. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science. 2022, 3756586): 1254-1261.

[81] Ufnalska I, Drew SC, Zhukov I, Szutkowski K, Wawrzyniak UE, Wroblewski W, Fraczyk T, Bal W. Intermediate Cu(II)-thiolate species in the reduction of Cu(II)GHK by glutathione: A handy chelate for biological Cu(II) reduction. Inorg Chem. 2021, 60(23): 18048-18057.

[82] Viles JH, Cohen FE, Prusiner SB, Goodin DB, Wright PE, Dyson HJ. Copper binding to the prion protein: structural implications of four identical cooperative binding sites. Proc Natl Acad Sci U S A. 1999, 96(5): 2042-2047.

[83] Villaverde A, Carrio MM. Protein aggregation in recombinant bacteria: Biological role of inclusion bodies. Biotechnol Lett. 2003, 25(17): 1385-1395.

[84] Waegeman H, Soetaert W. Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering. J Ind Microbiol Biotechnol. 2011, 38(12): 1891-1910.

[85] Wait R, James B, Calder MR. Synthesis and characterization by fast atom bombardment mass spectrometry of peptides related to the B‐domain of staphylococcal protein A. Org Mass Spectrom. 2005, 26(5): 458-462.

[86] Walsh G, Walsh E. Biopharmaceutical benchmarks 2022. Nat Biotechnol. 2022, 40(12): 1722-1760.

[87] Wu QJ, Zhang TN, Chen HH, Yu XF, Lv JL, Liu YY, Liu YS, Zheng G, Zhao JQ, Wei YF, Guo JY, Liu FH, Chang Q, Zhang YX, Liu CG, Zhao YH. The sirtuin family in health and disease. Signal Transduct Target Ther. 2022, 7(1. 402

[88] Yee L, Blanch HW. Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli. Biotechnology. 1992, 1012): 1550-1556.

[89] Zajda J, Wadych E, Ogorek K, Drozd M, Matczuk M. Novel applications of CE-ICP-MS/MS: monitoring of antiaging GHK-Cu cosmetic component encapsulation in liposomes. Electrophoresis. 2024, 45(21–22): 1946-1954.

[90] Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, Gold DS, Heinsohn HG, Murray CJ. Microscale to manufacturing scale-up of cell-free cytokine production: A new approach for shortening protein production development timelines. Biotechnol Bioeng. 2011, 108(7): 1570-1578.

[91] Zhai Y, Li O, Qi R, Zhang H, Ji XK, Li H. Method for preparing cysteine-modified polypeptides under high-density fermentation conditions, China. 2022.

[92] Zhang H, Wang Y, Lian L, Zhang C, He Z. Glycine-Histidine-Lysine (GHK) alleviates astrocytes injury of intracerebral hemorrhage via the Akt/miR-146a-3p/AQP4 pathway. Front Neurosci. 2020, 14. 576389

[93] Zhao C, Li D, Li J, Wang L. Molecular tandem repeat strategy for production ofultrashort peptides. Sheng Wu Gong Cheng Xue Bao. 2022, 38(12): 4587-4600.

[94] Zhou M, Wu X, Luo J, Yang G, Lu Y, Lin S, Jiang F, Zhang W, Jiang X. Copper peptide-incorporated 3D-printed silk-based scaffolds promote vascularized bone regeneration. Chem Eng J. 2021, 422. 130147

Funding

Key Technologies Research and Development Program of Anhui Province(2021YFC2100300)

National Natural Science Foundation of China(31871740)

RIGHTS & PERMISSIONS

Jiangnan University