Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.

Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.

Bordetella pertussis tracheal cytotoxin and other muramyl peptides: distinct structure-activity relationships for respiratory epithelial cytopathology.

Tracheal cytotoxin (TCT) is a disaccharide-tetrapeptide released by Bordetella pertussis, the causative agent of pertussis (whooping cough). We have previously determined the structure of TCT to be GlcNAc-1,6-anhydro-MurNAc-L-Ala-gamma-D-Glu-meso-A2pm-D-Ala, where MurNAc = N-acetylmuramic acid and A2pm = diaminopimelic acid. Purified TCT reproduces the respiratory cytopathology observed during pertussis, including ciliostasis and extrusion of ciliated cells. We have tested structural analogs of TCT for their ability to reproduce native TCT toxicity in explanted hamster tracheal tissue and hamster trachea epithelial (HTE) cell cultures. Other investigators have evaluated many of these analogs, which are muramyl or desmuramyl peptides, for muramyl peptide activities such as immunopotentiation, induction of slow-wave sleep, and pyrogenicity. Four desmuramyl peptides were produced in our laboratory from B. pertussis peptidoglycan or by chemical synthesis, including unusual peptides containing alpha-aminopimelic acid in place of A2pm. Based on the relative ability of compounds to inhibit DNA synthesis in HTE cells, truncated analogs lacking A2pm entirely or lacking only the side-chain amine or carboxyl group of A2pm were less active than TCT by a factor of at least 1000. All active analogs included a native or near-native peptide moiety, independent of the presence, absence, or substitution of the sugar moiety. We conclude that the structural requirements for TCT toxicity differ considerably from those for most other muramyl peptide activities, in that the disaccharide moiety is irrelevant for toxicity and both the free amino and carboxyl groups of the A2pm side chain are required for activity.

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.

Determination of the disulfide bond pairings in bovine transforming growth factor-alpha.

A rapid method for determining the three disulfide bond pairings in bovine transforming growth factor-alpha (bTGF-alpha) was developed by digesting bTGF-alpha with thermolysin followed by separation of the generated peptides by reversed-phase HPLC. The disulfide-bonded peptides were identified by amino acid sequencing and fast atom bombardment mass spectrometry. The disulfide bond pairings in bTGF-alpha were determined to be homologous to those in the human and mouse TGF-alpha molecules. A species of low bioactivity isolated from the folding/oxidation mixture of chemically synthesized bTGF-alpha was demonstrated to contain two incorrect disulfide bonds. These results indicate that mispairing of disulfide bonds in bTGF-alpha significantly reduces the activity of this molecule.