Functional expression of recombinant human ribonuclease/angiogenin inhibitor in stably transformed Drosophila melanogaster S2 cells.
A recombinant plasmid harboring heterologous genes coding human ribonuclease/angiogenin inhibitor (RAI) was expressed in stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Stably transformed polyclonal cell populations expressing RAI were isolated after 4 weeks of selection with hygromycin B. Recombinant RAI with a molecular weight of 50 kDa was detected in the intracellular (cell) and extracellular (medium) fractions of S2 cells. Recombinant RAI was purified from the extracellular fraction using a two-step purification scheme comprised of Ni-NTA and ion-exchange chromatography. Purified RAI migrated on SDS-PAGE as a single band in the elution fraction containing 300 mM NaCl. The ribonuclease inhibitor activity of purified RAI was measured using yeast tRNA and RNase A. Purified RAI exhibited an activity of approximately 8 U mug(-1) for the inhibition of RNA degradation by RNase A. Cultivation of stably transformed S2 cells using HyQ((R))SFX-insect MP medium increased cell growth by 79% and approximately doubled the production of recombinant RAI.
Park
Jong-Hwa
JH
Department of Advanced Technology Fusion and Bio/Molecular Informatics Centers, Konkuk University, Seoul, 143-701, South Korea.
Hwang
In-Sook
IS
Kim
Kyung-Il
KI
Lee
Jong-Min
JM
Park
Young-Min
YM
Park
Chang-Ho
CH
Chung
In Sik
IS