{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T04:14:19Z","timestamp":1788322459563,"version":"build-2803163510"},"reference-count":70,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,8,19]],"date-time":"2020-08-19T00:00:00Z","timestamp":1597795200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Science Technology, Taiwan","award":["MOST 105-2313-B-033-001-MY3 and MOST 107-2622-B-033-001-CC2 to C.-D.H. and MOST108-2113-M153-003 to K.H.-C.C."],"award-info":[{"award-number":["MOST 105-2313-B-033-001-MY3 and MOST 107-2622-B-033-001-CC2 to C.-D.H. and MOST108-2113-M153-003 to K.H.-C.C."]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>In this study, an alternative method is developed to replace chemical synthesis to produce glycyl-histidyl-lysine (GHK) tripeptides with a bacterial fermentation system. The target GHK tripeptides are cloned into expression plasmids carrying histidine-glutathione-S-transferase (GST) double tags and TEV (tobacco etch virus) cleavage sites at the N-terminus. After overexpression in Escherichia coli (E. coli) BL21 cells, the recombinant proteins are purified and recovered by high-pressure liquid chromatography (HPLC). UV-vis absorption spectroscopy was used to investigate the chemical and biological properties of the recombinant GHK tripeptides. The results demonstrated that one recombinant GHK tripeptide can bind one copper ion to form a GHK-Cu complex with high affinity, and the recombinant GHK peptide to copper ion ratio is 1:1. X-ray absorption near-edge spectroscopy (XANES) of the copper ions indicated that the oxidation state of copper in the recombinant GHK-Cu complexes here was Cu(II). All of the optical spectrum evidence suggests that the recombinant GHK tripeptide appears to possess the same biophysical and biochemical features as the GHK tripeptide isolated from human plasma. Due to the high binding affinity of GHK tripeptides to copper ions, we used zebrafish as an in vivo model to elucidate whether recombinant GHK tripeptides possess detoxification potential against the cardiotoxicity raised by waterborne Cu(II) exposure. Here, exposure to Cu(II) induced bradycardia and heartbeat irregularity in zebrafish larvae; however, the administration of GHK tripeptides could rescue those experiencing cardiotoxicity, even at the lowest concentration of 1 nM, where the GHK-Cu complex minimized CuSO4-induced cardiotoxicity effects at a GHK:Cu ratio of 1:10. On the other hand, copper and the combination with the GHK tripeptide did not significantly alter other cardiovascular parameters, including stroke volume, ejection fraction, and fractional shortening. Meanwhile, the heart rate and cardiac output were boosted after exposure with 1 nM of GHK peptides. In this study, recombinant GHK tripeptide expression was performed, along with purification and chemical property characterization, which revealed a potent cardiotoxicity protection function in vivo with zebrafish for the first time.<\/jats:p>","DOI":"10.3390\/biom10091202","type":"journal-article","created":{"date-parts":[[2020,8,19]],"date-time":"2020-08-19T09:22:31Z","timestamp":1597828951000},"page":"1202","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":21,"title":["Expression and Purification of Recombinant GHK Tripeptides Are Able to Protect against Acute Cardiotoxicity from Exposure to Waterborne-Copper in Zebrafish"],"prefix":"10.3390","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6398-8672","authenticated-orcid":false,"given":"Chung-Der","family":"Hsiao","sequence":"first","affiliation":[{"name":"Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320314, Taiwan"},{"name":"Master Program of Nanotechnology, Chung Yuan Christian University, Chung-Li 320314, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hsin-Hui","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry, National Pingtung University, Pingtung 900391, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nemi","family":"Malhotra","sequence":"additional","affiliation":[{"name":"Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320314, Taiwan"},{"name":"Department of Applied Chemistry, National Pingtung University, Pingtung 900391, Taiwan"},{"name":"Department of Biomedical Engineering, Chung Yuan Christian University, Chung-Li 320314, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yen-Ching","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry, National Pingtung University, Pingtung 900391, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying-Hsuan","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry, National Pingtung University, Pingtung 900391, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu-Nung","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320314, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ferry","family":"Saputra","sequence":"additional","affiliation":[{"name":"Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320314, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fiorency","family":"Santoso","sequence":"additional","affiliation":[{"name":"Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li 320314, Taiwan"},{"name":"Master Program of Nanotechnology, Chung Yuan Christian University, Chung-Li 320314, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelvin H.-C.","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Applied Chemistry, National Pingtung University, Pingtung 900391, Taiwan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,8,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"648108","DOI":"10.1155\/2015\/648108","article-title":"GHK peptide as a natural modulator of multiple cellular pathways in skin regeneration","volume":"2015","author":"Pickart","year":"2015","journal-title":"Biomed. 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