Preparative electrofocusing for industrial applications.

This paper presents a case study of using a multicompartment isoelectric focusing apparatus to determine the isoelectric points and focus preparative quantities of brain derived neurotrophic factor (BDNF) and neurotrophic factor 3 (NT3). A separation of PEGylated from unPEGylated forms of granulocyte colony stimulating factor (G-CSF) is described as well. Both BDNF and NT3 have substantially higher pI values in this system than is predicted from sequenced based modeling. Although PEGylated forms of G-CSF can be separated from the unPEGylated forms, separation of protein with differing degrees of PEGylation was not achieved. The paper additionally demonstrates that this technique can be used simultaneously as an analytical and preparative tool, eliminating the need for analytical IEF gels.

Strategies to suppress aggregation of recombinant keratinocyte growth factor during liquid formulation development.

Recombinant human keratinocyte growth factor (rhKGF) is a fairly unstable protein, posing a challenging problem for long-term storage. During storage, the protein unfolds at relatively low temperatures and the unfolded proteins aggregate rapidly, leading to the formation of large visible precipitates. Thermal unfolding of rhKGF displays a similar pattern, i.e., unfolding is followed immediately by aggregation as the temperature is increased. As the unfolding and aggregation (precipitation) of rhKGF limit the storage life of the protein, a search for stabilizers to suppress rhKGF unfolding and aggregation has been done by examining the effects of excipients on thermal melting temperature and on the rate of protein aggregation during storage. Sulfated polysaccharides and citrate are found to be effective in increasing the melting temperature of rhKGF or preventing its aggregation. In particular, 0.5% (w/v) heparin and high molecular weight dextran sulfate, and 0.5 M citrate are highly effective, decreasing the rates of rhKGF aggregation by about 50-fold. Other negatively charged small ions, such as phosphate, also have moderate stabilizing effects on rhKGF. A mechanistic study of the aggregation pathway of rhKGF has led to a better understanding of the stabilizing effects of these molecules. Molecules which enhance rhKGF conformational stability are capable of effectively suppressing rhKGF aggregation.

Properties of glycosylated and non-glycosylated human recombinant IGF binding protein-3 (IGFBP-3).

The interaction of insulin-like growth factor-I with glycosylated and nonglycosylated human recombinant IGFBP-3 was studied by competition binding and by realtime biospecific interaction analysis (BIA). No significant difference was found in the affinity of the two forms of recombinant IGFBP-3 for IGF-I. Combining the results from the two different methods of binding analysis, a dissociation constant of 50 +/- 14 pM was determined. This value compares favorably to that reported for IGF-I binding to natural human plasma-derived IGFBP-3. Subcutaneous injection into rats of IGF-I either bound to glycosylated or nonglycosylated IGFBP-3 yielded an area under the curve (AUC) that was essentially the same for either form of IGFBP-3 and was twice as large as the AUC obtained after injection of IGF-I in the free form. Circulating IGF-I peak levels were reached in less than 1 h post-injection for free IGF-I, 4 and 8 h post-injection for the nonglycosylated and glycosylated IGF-I/IGFBP-3 complexes respectively. Neutral gel filtration of rat serum samples 4 and 8 h post-injection revealed that the IGF-I delivered bound to the nonglycosylated IGFBP-3 circulated as a 40-50 kDa complex at the 4 h time point and as a 130-140 kD complex at the 8 h time point. IGF-I delivered as a complex with glycosylated IGFBP-3 was found to circulate as a 140 kD complex at 4 and 8 h post-injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of human recombinant insulin-like growth factor binding protein 3 expressed in Chinese hamster ovary cells.

Recombinant human insulin-like growth factor binding protein 3 (hIGFBP-3) stably expressed in chinese hamster ovary cells (CHO cells) has been purified to homogeneity from serum-free culture media. The purified protein migrates as a doublet (45/43 kDa) upon SDS-PAGE. The purified recombinant hIGFBP-3 is fully active and binds one mole of IGF-I per mole of recombinant binding protein. When the transfected CHO cells are treated with tunicamycin a single 29 kDa hIGFBP-3 protein is observed. This expressed hIGFBP-3 protein maintains its ability to bind IGF-I. N-Glycanase treatment of the purified hIGFBP-3 protein results in a protein that migrates similar to E. coli-derived IGFBP-3 upon SDS-PAGE under reducing conditions (30 kDa). Carboxymethylation of hIGFBP-3 suggests that all 18 cysteines are involved in disulfide linkages. These results represent the first purification and characterization of recombinant hIGFBP-3 expressed in CHO cells.

Human plasma fibronectin. Demonstration of structural differences between the A- and B-chains in the III CS region.

Fibronectins are a class of cell-adhesion proteins produced from a single gene. The soluble plasma form is synthesized by hepatocytes and the insoluble cellular form by fibroblasts and other cell types. The proteins possess multiple binding domains for macromolecules including collagen, fibrin and heparin along with at least one cell-binding domain. Cellular as well as plasma fibronectins are dimers of similar but not identical polypeptides. Their differences are the result of internal amino acid sequence variability due to alternative RNA splicing in at least three regions (ED-A, ED-B and III CS). We have been studying this polymorphism at the protein level in plasma fibronectin (pFn). Cathepsin D-digested pFn applied to a heparin-agarose column and eluted with an NaCl stepwise gradient (0.1 M, 0.25 M and 0.5 M) released two polypeptides (75 kDa and 65 kDa) in the 0.5 M-NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the C-terminal heparin-binding domain) and AHB-3 (specific for the III CS domain) suggest that both peptides contain the C-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the III CS region. These two polypeptides (75 kDa and 65 kDa) were digested with trypsin, and the resulting peptides were analyzed by fast-atom-bombardment mass spectrometry and compared with the known cDNA-derived peptide sequence. Peptides that were unique to the III CS region were further characterized by micro sequence analysis. The 75 kDa fragment is derived from the A-chain and contains the III CS region (89 amino acid residues) along with the C-terminal heparin-binding (Hep-2) domain and the fibrin-binding (Fib-2) domain. A single galactosamine-based carbohydrate group was detected at Thr-73/74 of the III CS region present in the 75 kDa fragment. The 65 kDa fragment is derived from the B-chain and lacks the entire III CS region but does contain the Hep-2 and Fib-2 domains.

Altered expression of fibronectin gene in cells infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) induces morphological changes in infected cells that are remarkably similar to those seen in oncogenically transformed cells. The molecular bases of these phenotypic alterations are not known but their occurrence in some transformed cells can be associated with abnormal fibronectin (FN) expression. In this report, we have compared FN levels in normal and HCMV-infected cells. In these studies, the HCMV-infected fibroblasts exhibited a progressive loss of cellular FN. Northern (RNA) blot analysis revealed that the decrease in FN levels resulted from a lowering of FN mRNA levels in HCMV-infected cells. We detected an initial decrease in FN mRNA of 25 to 30% at immediate-early and early times, whereas at late times after infection the levels of FN mRNA were lowered by greater than 80%. These results indicated that the HCMV-induced decrease in FN expression is due to a decrease in the quantity of FN mRNA and suggested that HCMV-encoded and/or -induced functions may be involved in producing these alterations.

Human placental fibronectin: demonstration of structural differences between the A and B chains in the extra domain-A region.

Fibronectins are a class of cell adhesion proteins produced from a single gene. Soluble plasma fibronectin plays a role in wound healing and the insoluble cellular fibronectin form anchors cells to the substrata. The proteins possess multiple macromolecular binding domains including collagen, fibrin, and heparin. Alternative RNA splicing in at least three regions (ED-A, ED-B, and III CS) is responsible for this fibronectin polymorphism. We have been studying this polymorphism at the protein level in placental fibronectin, a poorly soluble form of cellular fibronectin. Cathepsin D-digested placental fibronectin applied to a heparin-agarose column and eluted with a NaCl stepwise gradient (0.1, 0.3, 0.5 M) gave two polypeptides (80-100 and 65 kDa) in the 0.3 M NaCl peak. Immunoblots with monoclonal antibodies IST-2 (specific for the carboxy-terminal heparin-binding domain) and IST-9 (specific for the ED-A portion of fibronectin) suggest that both peptides contain the carboxy-terminal heparin-binding (Hep-2) domain, but that only the larger fragment possesses the ED-A segment. The two peptides were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to Polybrene-coated polyvinyl difluoride membranes, and characterized by microsequence analysis. This analysis confirmed that both fragments start with the same amino acid sequence, 17 amino acids before the start of ED-A. These results demonstrate that placental fibronectin is a heterodimer, structurally distinct from plasma fibronectin due to the presence of a unique domain modification that is not seen in the plasma form.

Interaction between L-threonine dehydrogenase and aminoacetone synthetase and mechanism of aminoacetone production.

A mixture of threonine dehydrogenase and aminoacetone synthetase will catalyze the conversion of L-threonine to glycine. The overall reaction likely involves the conversion of L-threonine, NAD+, and CoA to glycine, NADH, and acetyl-CoA. Physical separation of L-threonine dehydrogenase from aminoacetone synthetase results in the formation of aminoacetone and CO2 from their substrates. A physical interaction between threonine dehydrogenase and aminoacetone synthetase has been demonstrated by gel permeation chromatography and fluorescence polarization. Polarization of fluorescence measurements of threonine dehydrogenase and aminoacetone synthetase labeled with fluorescein isothiocyanate indicated the formation of a soluble active complex, with an apparent dissociation constant (Kd) of 5-10 nM and an apparent stoichiometry of 2 aminoacetone synthetase dimers/1 threonine dehydrogenase tetramer. Chemical experiments have identified aminoacetone as the enzymatic product of L-threonine dehydrogenase acting on L-threonine. These experiments involved trapping pyrrole derivatives, [3H]NaBH4 reduction, and coupling with plasma amine oxidase. Kinetic experiments also showed NADH, CO2, and aminoacetone to inhibit threonine dehydrogenase in a manner consistent with an ordered Bi-Ter kinetic mechanism. NAD+ is the lead substrate followed by threonine, and the products are released in the order: CO2, aminoacetone, and NADH.

Purification and properties of aminoacetone synthetase from beef liver mitochondria.

Aminoacetone synthetase from beef liver mitochondria was purified to homogeneity and shown to be a member of the pyridoxal 5'-phosphate-dependent family of enzymes. This enzyme catalyzes the condensation of glycine and acetyl-CoA to produce CO2, CoA, and the stable product aminoacetone. Bovine aminoacetone synthetase is a dimer (Mr 56,000) of identical subunits and contains 2 mol of pyridoxal phosphate/mol of dimer. The holoenzyme was resolved by dialysis against cysteine and has a pI of 5.2. The holoenzyme shows an absorption maximum at 428 nm which undergoes a shift to 335 nm when reduced with sodium borohydride. The Km values of glycine and acetyl-CoA were 22 mM and 53 microM, respectively. Initial velocity studies indicate that the condensation reaction proceeds by an ordered mechanism. With the exception of aminomalonate, bovine aminoacetone synthetase acts specifically on glycine and acetyl-CoA. Coupled reactions of purified bovine aminoacetone synthetase and porcine L-threonine dehydrogenase demonstrated the interconversion of threonine and glycine.