Masaharu Seno

Stabilizing basic fibroblast growth factor using protein engineering

Using site directed mutagenesis, each of the four cysteines present at amino acid residues 26, 70, 88, and 93 of the mature protein of human basic fibroblast growth factor (bFGF) was individually changed to serine. The biological activity and heparin binding ability was retained when the serine was substituted for the cysteine residue at either 70 or 88 of the bFGF protein. This finding indicates that the cysteines at these positions are not essential for expressing biological activity. The substitution of the residues at these positions, especially at position 88, reduced the heterogeneity recognized as several peaks of bFGF eluted from a heparin affinity column, even after oxidation with hydrogen peroxide, suggesting that the cysteines at these positions are exposed to the surface of the molecule to form disulfide bonds that induce heterologous conformations. Furthermore, under acidic conditions, these modified bFGFs are revealed to be more stable in maintaining their activity. These facts suggest that this protein has been successfully modified by protein engineering.

Expression of cDNA encoding human basic fibroblast growth factor in E. coli

The cDNA encoding human basic fibroblast growth factor was expressed in E. coli under the control of trp promoter. Bacterially synthesized hbFGF was highly purified using a heparin affinity HPLC column. By this chromatography, hbFGF was eluted as four distinct forms, which were indistinguishable by SDS polyacrylamide gel electrophoresis, amino acid composition, and partial terminal sequence analysis. These molecules stimulated the growth of fibroblasts and endothelial cells although their specific activities varied. The angiogenesis activity of these molecules was also confirmed.

A hybrid protein between IFN‐γ and IL‐2

The complementary DNAs encoding human interferon‐γ (IFN‐γ) and human interleukin‐2 (IL‐2), two different proteins involved in the same immune system, were fused to code a hybrid protein, which was expressed in E. coli to investigate the interactions of these two proteins at the molecular level. Through immunoprecipitation analysis, this protein was revealed to be of about 31 kDa, which was expected from nucleotide sequencing, and to have the antigenicities of both IFN‐γ and IL‐2. The extract from bacteria expressing this hybrid protein showed at least two biological activities: an antiviral activity derived from IFN‐γ and the ability to support the growth of natural killer (NK) cells derived from IL‐2. Comparing the enhancement of NK cell activity of this hybrid protein with IFN‐γ and IL‐2, this hybrid protein appears to conserve each activity almost completely without diminishing the other.

Two cDNAs encoding novel human FGF receptor

Two types of cDNAs encoding novel human FGF receptors were isolated. These two cDNAs were found to be closely related to the oncogene bek. Products from these genes were membrane-bound when their cDNAs were transiently expressed in COS cells, whereas products from the regions coding extracellular domains were free of membrane attachment and found in the culture medium.

Synthesis of hepatitis B virus e antigen in E. coli

Hepatitis B virus core antigen (HBcAg) gene was deleted at some unique restriction enzyme sites, or at random, and inserted into the expression plasmids of E. coli which had the tryptophan promoter. E. coli transformants with the plasmids, synthesized materials with many kinds of antigenicity of HBcAg, HBeAg, or both HBcAg and HBeAg. HBeAg-specific material smaller than native HBeAg was produced in a stable condition.

Establishment of monoclonal antibodies against human acidic fibroblast growth factor

Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four MAbs designated AF1-52, 81, 114 and 1C10 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AF1-52, 114, and 1C10 MAbs recognize epitopes within the 1-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF1-81 MAb could not be determined. The sandwich EIA method constructed with these MAbs was sensitive to 1.5 pg/well of haFGF and had no cross-reactivity with human basic FGF, bovine aFGF or the hst-1 gene product.