Extraction of endoglucanase I (Ce17B) fusion proteins from Trichoderma reesei culture filtrate in a poly(ethylene glycol)-phosphate aqueous two-phase system.
Endoglucanases (EGI) (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Ce17B) of Trichoderma reesei are industrially important enzymes. Thus, there is a great need for development of a primary recovery method suitable for large-scale utilization. In this study we present a concept applicable for large-scale purification of an EGI fusion protein by one-step extraction in a poly(ethylene glycol) PEG-sodium/potassium phosphate aqueous two-phase system. EGI is a two-module enzyme composed of an N-terminal catalytic module and a C-terminal cellulose binding module (CBM) separated by a glycosylated linker region. Partitioning of six different EGI constructs, containing the C-terminal extensions (WP)2, (WP)4 or the amphiphilic protein hydrophobin I (HFB) of T. reesei instead of the CBM were studied to evaluate if any of the fusions could improve the partition coefficient sufficiently to be suitable for large-scale production. All constructs showed improved partitioning in comparison to full length EGI. The (WP)4 extensions resulted in 26- to 60-fold improvement of partition coefficient. Consequently, a relative minor change in amino acid sequence on the two-module protein EGI improved the partition coefficient significantly in the PEG 4000-sodium/potassium phosphate system. The addition of HFBI to EGI clearly enhanced the partition coefficient (K=1.2) in comparison to full-length EGI (K=0.035). Partitioning of the construct with (WP)4 fused to the catalytic module and a short sequence of the linker [EGI(core-P5)(WP)4] resulted in the highest partition coefficient (K=54) and a yield of 98% in the PEG phase. Gel electrophoresis showed that the construct with the (WP)4 tag attached after a penta-proline linker could be purified from the other bulk proteins by only a single-step separation in the PEG 4000-sodium/potassium phosphate system. This is a major improvement in comparison with the previously studied model (ethylene oxide-propylene oxide)-dextran system. Hence, this construct will be suitable for further optimization of the extraction of the enzyme in a PEG 4000-sodium/potassium phosphate system from culture filtrate.
Collén
Anna
A
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden.
Penttilä
Merja
M
Stålbrand
Henrik
H
Tjerneld
Folke
F
Veide
Andres
A
D002480:Cellulase / Q000302:isolation & purification* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D004591:Electrophoresis, Polyacrylamide Gel; D010710:Phosphates / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014242:Trichoderma / Q000737:chemistry / Q000201:enzymology*; D014867:Water / Q000737:chemistry
D010710:Phosphates; D011993:Recombinant Fusion Proteins; D014867:Water; D011092:Polyethylene Glycols; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase