The present paper describes the total chemical synthesis of the precursor molecule of the Aequorea green f luorescent protein (GFP). The molecule is made up of 238 amino acid residues in a single polypeptide chain and is nonf luorescent. To carry out the synthesis, a procedure, first described in 1981 for the synthesis of complex peptides, was used. The procedure is based on performing segment condensation reactions in solution while providing maximum protection to the segment. The effectiveness of the procedure has been demonstrated by the synthesis of various biologically active peptides and small proteins, such as human angiogenin, a 123-residue protein analogue of ribonuclease A, human midkine, a 121-residue protein, and pleiotrophin, a 136-residue protein analogue of midkine. The GFP precursor molecule was synthesized from 26 fully protected segments in solution, and the final 238-residue peptide was treated with anhydrous hydrogen f luoride to obtain the precursor molecule of GFP containing two Cys(acetamidomethyl) residues. After removal of the acetamidomethyl groups, the product was dissolved in 0.1 M Tris⅐HCl buffer (pH 8.0) in the presence of DTT. After several hours at room temperature, the solution began to emit a green f luorescence ( max ؍ 509 nm) under near-UV light. Both f luorescence excitation and f luorescence emission spectra were measured and were found to have the same shape and maxima as those reported for native GFP. The present results demonstrate the utility of the segment condensation procedure in synthesizing large protein molecules such as GFP. The result also provides evidence that the formation of the chromophore in GFP is not dependent on any external cofactor.The bioluminescent jellyfish Aequorea victoria produces a greenish luminescence from the margin of its umbrella by using two proteins: a Ca 2ϩ -binding protein, aequorin (21.4 kDa) and a chromophore-bearing green fluorescent protein (GFP; 27 kDa) (1). On binding Ca 2ϩ , aequorin undergoes an intramolecular reaction, yielding a blue fluorescent protein in the singlet excited state, which transfers its energy by resonance to GFP. The acceptor of energy in GFP is a chromophore consisting of an imidazolone ring structure (2-4). A phenolate anion of the chromophore in the singlet excited state is the emitter of the green light (4). Chemical studies of GFP and expression of cDNA for GFP in prokaryotes and eukaryotes have shown that the chromophore is a product of a posttranslational modification of the primary structure, involving Aequorea GFP consists of 238 amino acid residues in a single polypeptide chain (7-9). The native molecule has been shown to regenerate its intrinsic fluorescence from the totally denatured state (10). GFP is a relatively large molecule to serve as a target for chemical synthesis because a 99-residue HIVprotease analogue is the largest protein synthesized thus far in a highly homogeneous form by the solid-phase method (11) and the 136-residue human pleiotrophin (12) is the largest protein that h...
Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.
Human midkine (hMK), a novel heparin-binding neurotrophic factor consisting of 121 amino acid residues with five intramolecular disulphide bonds, was synthesized by solution procedure in order to demonstrate the usefulness of our newly developed solvent system, a mixture of dichloromethane or chloroform and trifluoroethanol. The final protected 121-residue peptide was assembled from two large fully protected intermediates, Boc-(1-59)-OH and H-(60-121)-OBzl, in CHL/TFE(3:1, v/v) using water-soluble carbodiimide in the presence of HOOBt as coupling reagents. After removal of the protecting groups by HF followed by treatment with Hg(OAc)2 in 50% acetic acid, the fully deprotected peptide was subjected to the oxidative folding reaction. The final product was confirmed to have the correct disulphide structure from its tryptic peptide mapping and to possess the same biological activities as those of the natural product. In order to clarify the active region of the hMK molecule, the N-terminal half domains [(1-59) and (60-121)] were also synthesized by the same procedure used for the hMK synthesis. The C-half domain was confirmed to show the full pattern of bioactivities except for the neuronal cell survival activity, while the N-half one showed much less activity in general.
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