Selective release of recombinant protein particles (VLPs) from yeast using a pure lytic glucanase enzyme.
We have used a pure lytic glucanase enzyme to selectively release recombinant 60 nm protein particles (virus like particles or VLPs) from yeast cells. Although the protease components of the lytic enzyme complexes were found to degrade the VLPs, purified glucanase enzymes from these complexes (derived from Cytophaga sp. and Oerskovia sp.) produced cell lysis without degradation and released the VLPs in the absence of mercaptoethanol, a reducing agent commonly used in cell lysis. The Oerskovia glucanase enzyme released the recombinant protein particles selectively as it only produced ca. 17% cell lysis compared to the use of the total lytic enzyme preparation. The use of osmotic supports did not improve the recovery of VLPs, however, treatment of the enzymatically lysed pellet with Triton X-100 did increase the amount of VLPs released. This 'selectivity', which results in the release of the recombinant particles with only a fraction of contaminating proteins, represents an improvement over presently used mechanical or enzymatic cell disruption processes.
Asenjo
J A
JA
Biochemical Engineering Laboratory, University of Reading, England.
Ventom
A M
AM
Huang
R B
RB
Andrews
B A
BA
D000192:Actinomycetales / Q000201:enzymology; D003589:Cytophaga / Q000201:enzymology; D004251:DNA Transposable Elements; D006026:Glycoside Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification*
D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases