Determination of Dimeric Disulfide Linkage in a Recombinant Human Prolactin Antagonist
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Date
2005-06
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The Ohio State University
Abstract
ΔhPRL is a potent human prolactin (hPRL) antagonist, engineered by the deletion of 12 key residues (residues 41 through 52) from wild-type hPRL. The resulting monomeric protein displays potent antagonist activity with little remaining agonist activity, however an undesirable result of this deletion is difficulty in folding, as evidenced by significant formation of dimeric species. The dimeric species is biologically inert. ΔhPRL is derived from wild-type hPRL a 199-residue single peptide protein containing three native disulfide bonds (C4--C11, C58--C174, C191--C199). Using mass spectrometric and additional biochemical techniques, we intend to determine the nature of the linkage found in dimeric ΔhPRL and identify key residues participating in this linkage.
Recombinant ΔhPRL, methionyl protein expressed using BL-21 E. coli, was folded, and purified from cell lysates by ion-exchange chromatography. Monomeric and dimeric species were separated by size-exclusion chromatography. Both species were characterized by absorbance spectroscopy, luminescence spectroscopy, and intact accurate mass determination by electro-spray Q-TOF. Each sample was analyzed by SDS-PAGE under reducing (2-BME) and non-reducing conditions. Dimeric ΔhPRL was denatured in urea, alkylated, and digested with trypsin. Tryptic peptides were analyzed by MALDI-TOF and electrospray LC/MS/MS to generate peptide maps and sequence information.
Accurate mass determination of intact ΔhPRL revealed that the molecular weight of dimeric species is twice the molecular weight of monomeric species, indicating that dimer is made of two constituent monomers with no sequence modification. Comparison of reducing and non-reducing SDS-PAGE analyses shows that dimeric species present after folding are resolved into purely monomeric constituents when treated with 2-BME, implying that dimeric linkage is due to inter-molecular disulfide bond formation. Further, spectroscopic variation between monomeric and dimeric species indicates that folding is perturbed in the dimeric species, increasing the prospect of opportunistic inter-molecular disulfide formation between cysteine residues. Peptide maps with approximately 70% coverage and sequence data resulting from tryptic digestion of alkylated dimeric ΔhPRL display cysteine-containing peptides in a mixture of disulfide-linked and free (alkylated) states, suggesting random intra-molecular and inter-molecular pairing between available cysteines. Observation of the disulfide-linked peptide 11-16 paired to 11-16 implies C11--C11 disulfide formation. This disulfide can only exist between molecules, suggesting that dimeric linkage is the product of intermolecular disulfide bonding between C11 residues.
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Keywords
Biochemistry, Prolactin, Disulfide, MS/MS, Digestion