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Production of a discrete, heterogeneous population of β‐galactosidase polypeptides using baculovirus expression vectors
Author(s) -
Licari P.,
Bailey J. E.
Publication year - 1992
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.260390908
Subject(s) - polyhedrin , biology , population , microbiology and biotechnology , transcription (linguistics) , fusion protein , heterologous , spodoptera , protein biosynthesis , baculoviridae , heterologous expression , autographa californica , recombinant dna , biochemistry , gene , linguistics , philosophy , demography , sociology
Gel electrophoresis analysis of immunoprecipitated β‐galactosidase and polyhedrin‐β‐galactosidase expressed in Spodoptera frugiperda cells infected with recombinant Autograph californica nuclear polyhedrosis virus revealed the existence of a population of discrete β‐galactosidase polypeptides. Several of the polypeptides observed in the fusion protein expression experiments exhibit a consistent pattern of slightly greater molecular weight when compared to the nonfusion β‐galactosidase that is compatible with the hypothesis that these fusion protein fragments retain the N‐terminal polyhedrin residues. Pulse‐chase experiments showed that overall β‐galactosidase degradation occurred at a negligible rate compared to the synthesis rate at 96 h postinfection, yet the fragments are observed for short pulse times. Degradation of several different β‐galactosidase polypeptides was observed 24 h postinfection. Ribonucleic acid hybridization analysis of lacZ transcripts shows significant heterogeneity that may result from premature transcription termination. Although a proteolytic origin cannot be excluded, the data assembled suggest that premature termination of transcription or translation is the likely cause for the heterogeneous population of immunoreactive peptides observed. Many discrete forms of β‐galactosidase polypeptides were also observed in studies with Escherichia coli , indicating that production of these heterogeneous forms is not a consequence of heterologous expression of the enzyme.