Identification of the bioactive peptide PEC-60 in brain.

PEC-60 is a 60-residue peptide originally isolated from pig intestine. It inhibits glucose-induced insulin secretion from perfused pancreas in a hormonal manner and also has biological activity in the immune system. PEC-60-like immunoreactive material has been reported in catecholamine neurons of the central and peripheral nervous systems, but the peptide has not been identified from that material. We have now isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is expressed in the gastrointestinal tract and the central nervous system. The specific regional brain distribution and interaction with classical neurotransmitters raise the possibility that PEC-60 may play a role in the central nervous system disorders involving dopamine dysregulation.

Molecular characterization of native and recombinant apolipoprotein A-IMilano dimer. The introduction of an interchain disulfide bridge remarkably alters the physicochemical properties of apolipoprotein A-I.

The disulfide-linked dimer of apolipoprotein A-IMilano (A-IM/A-IM), a natural Arg173-->Cys variant of apoA-I, was purified from carriers' plasma and produced in Escherichia coli. The recombinant A-IM/A-IM is identical to native A-IM/A-IM, by mass spectrometry, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing. Lipid-free A-IM/A-IM undergoes concentration-dependent self-association similar to apoA-I, but at all concentrations apoA-I is more self-associated than A-IM/A-IM. Far-ultraviolet CD spectra of A-IM/A-IM reveal a highly alpha-helical structure predicted to be approximately 65% in the lipid-free and approximately 78% in the lipid-associated states, versus 43 and 73% for apoA-I. A significant loss of alpha-helix occurs below pH 3.5 and above pH 10 in both apoA-I and A-IM/A-IM; A-IM/A-IM constantly shows a higher alpha-helical content than apoA-I over the entire pH range (1.7-12.8), suggesting that hydrophobic forces stabilize the interaction between the two A-IM chains. Indeed, and differently from apoA-I, the alpha-helical content of A-IM/A-IM is minimally affected by solvent ionic strength. The aromatic side chains in both lipid-free and lipid-bound A-IM/A-IM are immobilized in a more asymmetric and hydrophobic environment than in lipid-free apoA-I, the conformation of A-IM/A-IM being instead similar to that achieved by apoA-I following interaction with lipids. The present findings prove that rA-IM/A-IM is structurally identical to the native protein; the conformation of A-IM/A-IM is remarkably different from that of apoA-I, thus possibly explaining some of the peculiar functional properties of the apoA-IMilano dimer.

Purification and characterization of recombinant human insulin-like growth factor II (IGF-II) expressed as a secreted fusion protein in Escherichia coli.

Human insulin-like growth factor II (IGF-II) was produced in an Escherichia coli ompT strain as a 22.5-kDa fusion protein. IGF-II was fused to the carboxy-terminal of a synthetic 15-kDa IgG-binding protein, originating from staphylococcal protein A, via a unique methionine linker. During fermentation, the fusion protein was exported to the growth medium at levels exceeding 900 mg/liter and subsequently affinity purified on IgG Sepharose followed by ion exchange on S Sepharose. After chemical cleavage with CNBr, yielding an authentic IGF-II molecule, the recombinant IGF-II was purified to homogeneity by a two step procedure involving ion-exchange and reverse-phase HPLC. A substantial fraction of the secreted protein was found to be biologically active, eliminating the need for complex refolding procedures. The yield of highly purified and biologically active IGF-II was 5-7 mg/liter of fermenter broth. The IGF-II produced by this method displayed biochemical, immunological, receptor binding, and biological activity properties equal to those of native IGF-II isolated from human serum.

Characterization of an extended form of recombinant human insulin-like growth factor II.

To investigate the biological role of variants of human insulin-like growth factor II (IGF-II), an extended form designated IGF-IIE21, with a molecular mass of 9.8 kDa, was produced in Escherichia coli as a stable and soluble secreted fusion protein. After site-specific cleavage of the affinity purified fusion protein, followed by purification using ion exchange and reversed phase chromatography, it could be demonstrated that IGF-IIE21 and IGF-II have similar or identical activities according to radioimmunoassay and radioreceptor assay. However, IGF-IIE21 showed only 1% growth promotion activity as compared with IGF-II in a clonal expansion assay using human K562 cells which lacks IGF-I receptors. These results suggest that this extended variant of IGF-II can bind to the receptor but has limited growth promoting activity.

Facilitated in vitro refolding of human recombinant insulin-like growth factor I using a solubilizing fusion partner.

We describe a new approach to refolding recombinant proteins in which an affinity fusion partner, consisting of two IgG-binding domains (ZZ) derived from staphylococcal protein A, is used to solubilize misfolded molecules before, during and after reduction and reoxidation. We show that human insulin-like growth factor I (IGF-I) can be refolded as a fusion protein at a concentration as high as 1-2 mg/ml without the use of denaturing agents. A process scheme suitable for large scale application is described in which the yield of correctly folded human IGF-I with full biological activity is substantially increased.