NDLI: Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast

Content Provider	Springer Nature Link
Author	Wan, Wen Wang, Dongmei Gao, Xiaolian Hong, Jiong
Copyright Year	2011
Abstract	Easy and low-cost protein purification methods for the mass production of commonly used enzymes that play important roles in biotechnology are in high demand. In this study, we developed a fast, low-cost recombinant protein purification system in the methylotrophic yeast Pichia pastoris using the family 3 cellulose-binding module (CBM3)-based affinity tag. The codon of the cbm3 gene from Clostridium thermocellum was optimized based on the codon usage of P. pastoris. The CBM3 tag was then fused with enhanced green fluorescent protein (CBM3-EGFP) or with inulinase and expressed in P. pastoris to demonstrate its ability to function as an affinity tag in a yeast expression system. We also examined the effects of glycosylation on the secreted CBM3-tag. The secreted wild-type CBM3-EGFP was glycosylated; however, this had little influence on the adsorption of the fusion protein to the regenerated amorphous cellulose (RAC; maximum adsorption capacity of 319 mg/g). Two CBM3-EGFP mutants lacking glycosylation sites were also constructed. The three CBM3-EGFPs expressed in P. pastoris and the CBM3-EGFP expressed in Escherichia coli all had similar RAC adsorption capacity. To construct a tag-free recombinant protein purification system based on CBM3, a CBM3-intein-EGFP fusion protein was expressed in P. pastoris. This fusion protein was stably expressed and the self-cleavage of intein was efficiently induced by DTT or l-cysteine. In this study, we were able to purify the recombinant fusion protein with high efficiency using both intein and direct fusion-based strategies.
Starting Page	789
Ending Page	798
Page Count	10
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	91
Issue Number	3
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2011-06-09
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Cellulose-binding module Affinity tag Yeast Glycosylation Microbiology Microbial Genetics and Genomics Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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