Primary recovery of a genetically engineered Trichoderma reesei endoglucanase I (Cel 7B) fusion protein in cloud point extraction systems.
Here we present data to demonstrate how partitioning of a hydrophilic enzyme can be directed to the hydrophobic detergent-enriched phase of an aqueous two-phase system by addition of short stretches of amino acid residues to the protein molecule. The target enzyme was the industrially important endoglucanase I, EGI (endo-1,4-beta-D-glucan-4-glucanohydrolase, EC 3.2.1.4, Cel7B) of Trichoderma reesei. We investigated the partitioning of three EGI variants containing various C-terminal peptide extensions including Trp-Pro motifs of different lengths and localizations. Additionally, a recently developed system composed of the thermoseparating copolymer HM-EOPO was utilized to study the effects of fusion tags. The addition of peptides containing tryptohan residues enhanced the partitioning of EGI to the HM-EOPO-rich phase. The system composed of a nonionic detergent (Agrimul NRE1205) resulted in the highest partition coefficient (K = 31) and yield (90%) with the construct EGI(core-P5)(WP)(4) containing (Trp-Pro)(4) after a short linker stretch. A recombinant strain of T. reesei Rut-C30 for large-scale production was constructed in which the fusion protein EGI(core-P5)(WP)(4) was expressed from the strong promoter of the cellulase gene cbh1. The fusion protein was successfully expressed and secreted from the fungus during shake-flask cultivations. Cultivation in a 28-L bioreactor however, revealed that the fusion protein is sensitive to proteases. Consequently, only low production levels were obtained in large-scale production trials.
Collén
Anna
A
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-221 00, Sweden.
Selber
Klaus
K
Hyytiä
Teppo
T
Persson
Josefine
J
Nakari-Setlä
Tiina
T
Bailey
Michael
M
Fagerström
Richard
R
Kula
Maria-Regina
MR
Penttilä
Merja
M
Stålbrand
Henrik
H
Tjerneld
Folke
F
D019149:Bioreactors; D015153:Blotting, Western; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D002623:Chemistry Techniques, Analytical / Q000379:methods; D003902:Detergents / Q000737:chemistry; D005285:Fermentation; D057927:Hydrophobic and Hydrophilic Interactions; D010865:Pilot Projects; D011095:Polyethylenes / Q000737:chemistry; D011126:Polypropylenes / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity; D013501:Surface-Active Agents / Q000737:chemistry; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics; D014364:Tryptophan / Q000302:isolation & purification / Q000378:metabolism
D003902:Detergents; D011095:Polyethylenes; D011126:Polypropylenes; D011993:Recombinant Fusion Proteins; D013501:Surface-Active Agents; D014364:Tryptophan; C078661:UCON 50-HB-5100; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase