Ikehara Morio

Expression of a synthetic human growth hormone gene in yeast

Abstract A synthetic human growth hormone (hGH) gene was efficiently expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). More than 106 molecules of hormone were formed per cell despite the fact that the gene was constructed with codon preference for Escherichia coli.

Expression of human lysozyme in an insoluble form in yeast

Abstract A high level of expression in yeast of a chemically synthesized human lysozyme (hL) gene was achieved by introducing an A-rich DNA fragment just upstream from the ATG start codon. The synthesized recombinant human lysozyme (r-hL) was insoluble and biologically inactive. It was solubilized with 7 M urea (pH 9) from yeast cells and its lytic activity was efficiently regenerated by oxidative renaturation. This rknaturation experiment and Western blotting analysis under reducing and non-reducing conditions indicate that the insoluble form might be caused by the formation of incorrect intra- or intermolecular disulfide bonds. The N-terminal amino acid sequence of the purified r-hL was identical with that of authentic hL.

Comparison of the antigenicities of native human growth hormone (hGH) and three forms of recombinant hGHs using monoclonal antibodies

A panel of hybridomas producing antibodies specific for human growth hormone (hGH) were prepared by using a recombinant hGH [methionylsomatotropin (r-hGH)] as an immunogen. Thirteen representative monoclonal antibodies which showed different reactivity patterns were used to analyze the antigenicities of four different forms of hGHs by RIA inhibition studies. Native hGH and r-hGH showed almost the same antigenicities with these monoclonal antibodies. A Cys-substituted recombinant hGH (r-hGH-165) retained the epitopes recognized by 11 monoclonals but not those recognized by two monoclonals. All except one of the monoclonals showed little or no reactivity with a recombinant hGH fragment (r-hGH-AB). On the basis of these results, the differences in the structures and antigenicities of the recombinant hGH proteins were discussed.

High-level secretion of the extracellular domain of the human growth hormone receptor using a baculovirus system

Abstract A chemically synthesized gene (hGHR-ED) coding for the extracellular domain (ED) of the human growth hormone (hGH) receptor (hGHR) was inserted into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the polyhedrin promoter. Spodoptera frugiperda cells infected with the recombinant virus secreted a protein with hGH-binding activity into the medium. The secreted 35-kDa protein was purified to near homogeneity. The purified protein exhibited a high binding affinity ( K d = 0.2—0.3 nM ) to hGH. The highest cell production capability was estimated at more than 10–20 μg hGHR-ED/ml of culture. The inhibition of the hGHR-ED secretion by treatment with tunicamycin suggests that glycosylation is important for secretion.