AbstractHuman matrix Gla protein (MGP) is a vitamin K–dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6‐kD protein (84 amino acids) containing five γ‐carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 μg/mL). Because of solubility problems, processing of a recombinantly expressed MGP‐fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid‐phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1‐Ala53 was synthesized on a derivatized resin yielding a C‐terminal thioester group. Peptide Cys54‐Lys84 was synthesized on Lys‐PAM resin yielding a C‐terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1–84‐polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI‐MS), representing the loss of two protons because of the formation of the Cys54‐Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+‐binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in α‐helicity at 0.2 mM CaCl2 that may explain the Ca2+‐dependent shift in high‐pressure liquid chromatography (HPLC)‐retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification.