Purification and In Vitro Translation of Penicillium verruculosum Cellulase mRNA

  • Kim, Jeong-Ho (Institute of Biotechnology, Chonnam National University) ;
  • Chung, Ki-Chul (Institute of Biotechnology, Chonnam National University) ;
  • Kang, Hyun-Sam (Department of Microbiology, Seoul National University) ;
  • Lee, Young-Kyu (Institute of Biotechnology, Chonnam National University)
  • Published : 1991.12.01

Abstract

Caboxymethyl cellulase (CMCase) I was purified from the induced culture filtrate of Penicllium verruculosum F-3 by ammonium sulfate precipitation, DEAE-Sephadex A-50 chromatography and Bio-gel P-150 filtration. The purified enzyme was assumed to be a glycoprotein consisting of 8.5% carbohydrate and having a molecular weight of 70.000 in SDS-polycrylamide gel electrophoresis (SDS-PAGE). The purified enzyme-specific anti-CMCase I IgG was obtained by rabbit immunization and protein A-sepharose CL-4B chromatography. The fungal poly($A^+$) RNA was isolated from the total RNA of the mycelium grown under cellulase induction conditions by oligo(dT)-cellulosse chromatography. The translation products in vitro were prepared by translating the isolated poly ($A^+$) RNA in rabbit reticulocyte lysate and analyzed by SDS-PAGE and fluorography. Of the translation products, CMCase I was identified by the immunoprecipitation against anti-CMCase I IgG.

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