Journal of Microbiology

Expression of human hypoxanthine phosphoribosyltransferase in Insect Cells Using a Baculovirus Vector: Lee, Chong Ho , Yang, Ji Won , Baek, Sang Ki , Yang, jai Myung; J. Microbiol. 1995;33(1):85-90.

Abstract: The hypoxanthine phosphoribosyltransferase (HPRT) catalyzes the conversion of hypoxanthine and guanine to the mononucleotide, IMP and GMP, respectively. For construction of recombinant AcNPV carrying human HPRT, a transfer vector p918 constructed by cloning full-length cDNA for human HPRT into pVL1393 and AnNPV genomic DNA were co-transfected into Sf21 cells. The tissue culture fluid containing extracellular virus was plaque assayed and a recombinant virus with occlusion minus phenotype was obtained by three rounds of plaque purification. Southern blot analysis and PRC results confirmed the insertion of the human HPRT cDNA within the recombinant virus(AcHPRT918). SDS-PAGE and Western blot analysis of the Sf21 cell extracts infected with AcHPRT918 indicated that human HPRT was expressed in insect cell. Large quantities of functional HPRT expressed in insect cells would facilitate characterization of the biological properties of this enzyme.

Oligomerization of the substitution mutants of autographa californica nuclear polyhedrosis Virus (AcNPV) gp64 glycoprotein: Kim, Ki Nam , Poo, Ha Ryoung , Yang, Jai Myung; J. Microbiol. 1997;35(1):72-77.

Abstract: The baculovirus gp64 glycoprotein is a major component of the envelope protein of budded virus (BV). It has been shown that the gp64 glycoprotein plays an essential role in the infection process, especialy fusion between virus envelope and cellular endosomic membrane. Recently we reported optimal conditions required for gp64-mediated membrane fusion in pGP64 DNA transfected Spodoptera frugiperda (Sf9) cells (H. J. Kim and J. M. Yang, Jour, Microbiology, 34, 7-14). In order to investigate the role of hydrophobicity within the fusion domain of the gp64 glycoprotein for membrane fusion, 13 mutants which have substitution mutation within hydrophobic region I were constructed by PCR-derived site-derected mutagenesis. Each mutated gp64 glycoproteins was transiently expressed by transfecting plasmid DNA into Spodoptera frugiperda (Sf9) cells. Oligomerization of the transisently expressed gp64 glycoproteins was a analysed by running them on SDS-polyacrylamide gel electrophoresis under non-reducing condition followed by immunoblotting. All of the mutant gp64 glycoproteins expect cysteine-228 were able to form trimers. These results suggest that hydrophobic region I of the gp64 may not be responsible for the oligomerization of the gp64 glycoprotein.

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