Skip to main content
SpringerLink
Log in

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

  • Methods and Protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Ahn J, Choi E, Lee H, Hwang S, Kim C, Jang H, Haam S, Jung J (2004) Enhanced secretion of Bacillus stearothermophilus L1 lipase in Saccharomyces cerevisiae by translational fusion to cellulose-binding domain. Appl Microbiol Biotechnol 64:833–839

    Article  CAS  Google Scholar 

  • Arnau J, Lauritzen C, Petersen GE, Pedersen J (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48(1):1–13

    Article  CAS  Google Scholar 

  • Babu KS, Antony A, Muthukumaran T, Meenakshisundaram S (2008) Construction of intein-mediated hGMCSF expression vector and its purification in Pichia pastoris. Protein Expres Purif 57(2):201–205. doi:10.1016/j.pep.2007.10.004

    Article  CAS  Google Scholar 

  • Boraston AB, McLean BW, Guarna MM, Amandaron-Akow E, Kilburn DG (2001a) A family 2a carbohydrate-binding module suitable as an affinity tag for proteins produced in Pichia pastoris. Protein Expres Purif 21(3):417–423

    Article  CAS  Google Scholar 

  • Boraston AB, Warren RAJ, Kilburn DG (2001b) Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis. Biochem J 358:423–430

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Chong S, Xu MQ (1997) Protein splicing of the Saccharomyces cerevisiae VMA intein without the endonuclease motifs. J Biol Chem 272(25):15587–15590

    Article  CAS  Google Scholar 

  • Chong S, Shao Y, Paulus H, Benner J, Perler FB, Xu MQ (1996) Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J Biol Chem 271(36):22159–22168

    Article  CAS  Google Scholar 

  • Elleuche S, Poggeler S (2010) Inteins, valuable genetic elements in molecular biology and biotechnology. Appl Microbiol Biot 87(2):479–489. doi:10.1007/s00253-010-2628-x

    Article  CAS  Google Scholar 

  • Esposito D, Chatterjee DK (2006) Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 17(4):353–358

    Article  CAS  Google Scholar 

  • Fong BA, Wu WY, Wood DW (2010) The potential role of self-cleaving purification tags in commercial-scale processes. Trends Biotechnol 28(5):272–279. doi:10.1016/j.tibtech.2010.02.003

    Article  CAS  Google Scholar 

  • Gong F, Sheng J, Chi ZM, Li J (2007) Inulinase production by a marine yeast Pichia guilliermondii and inulin hydrolysis by the crude inulinase. J Ind Microbiol Biotechnol 34(3):179–185. doi:10.1007/s10295-006-0184-2

    Article  CAS  Google Scholar 

  • Guerreiro CIPD, Fontes CMGA, Gama M, Domingues L (2008) Escherichia coli expression and purification of four antimicrobial peptides fused to a family 3 carbohydrate-binding module (CBM) from Clostridium thermocellum. Protein Expres Purif 59(1):161–168. doi:10.1016/j.pep.2008.01.018

    CAS  Google Scholar 

  • Hartley JL (2006) Cloning technologies for protein expression and purification. Curr Opin Biotechnol 17(4):359–366

    Article  CAS  Google Scholar 

  • Hong J, Tamaki H, Kumagai H (2007a) Cloning and functional expression of thermostable beta-glucosidase gene from Thermoascus aurantiacus. Appl Microbiol Biot 73(6):1331–1339

    Article  CAS  Google Scholar 

  • Hong J, Ye XH, Zhang YHP (2007b) Quantitative determination of cellulose accessibility to cellulase based on adsorption of a nonhydrolytic fusion protein containing CBM and GFP with its applications. Langmuir 23(25):12535–12540

    Article  CAS  Google Scholar 

  • Hong J, Wang YR, Ye XH, Zhang YHP (2008a) Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage. J Chromatogr A 1194(2):150–154

    Article  CAS  Google Scholar 

  • Hong J, Ye XH, Wang YR, Zhang YHP (2008b) Bioseparation of recombinant cellulose-bindning module-proteins by affinity adsorption on an ultra-high-capacity cellulosic adsorbent. Anal Chim Acta 621(2):193–199

    Article  CAS  Google Scholar 

  • Kavoosi M, Meijer J, Kwan E, Creagh AL, Kilburn DG, Haynes CA (2004) Inexpensive one-step purification of polypeptides expressed in Escherichia coli as fusions with the family 9 carbohydrate-binding module of xylanase 10A from T. maritima. J Chromatogr B 807:87–94

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Levy I, Shoseyov O (2002) Cellulose-binding domains biotechnological applications. Biotechnol Adv 20(3–4):191–213

    Article  CAS  Google Scholar 

  • Levy I, Shoseyov O (2004) Cross bridging proteins in nature and their utilization in bio- and nanotechnology. Curr Protein Pept Sc 5(1):33–49

    Article  CAS  Google Scholar 

  • Miller GO (1959) Use of dinitrosaiicyiic acid reagent for determination of reducing sugar. Anal Biochem 31:426–428

    CAS  Google Scholar 

  • Murashima K, Kosugi A, Doi RH (2003) Solubilization of cellulosomal cellulases by fusion with cellulose-binding domain of noncellulosomal cellulase engd from Clostridium cellulovorans. Proteins 50(4):620–628

    Article  CAS  Google Scholar 

  • Przybycien TM, Pujar NS, Steele LM (2004) Alternative bioseparation operations: life beyond packed-bed chromatography. Curr Opin Biotechnol 15:469–478

    Article  CAS  Google Scholar 

  • Ramirez C, Fung J, Miller RC, Antony R, Warren J, Kilburn DG (1993) A bifunctional affinity linker to couple antibodies to cellulose. Nat Biotech 11(12):1570–1573

    Article  CAS  Google Scholar 

  • Shoseyov O, Shani Z, Levy I (2006) Carbohydrate binding modules: biochemical properties and novel applications. Microbiol Mol Biol R 70(2):283–295

    Article  CAS  Google Scholar 

  • Tomme P, Boraston AB, McLean B, Kormos JM, Creagh AL, Sturch K, Gilkes NR, Haynes CA, Warren RA, Kilburn DG (1998) Characterization and affinity applications of cellulose-binding domains. J Chromatogr B Biomed Sci Appl 715:283–296

    Article  CAS  Google Scholar 

  • Tormo J, Lamed R, Chirino AJ, Morag E, Bayer EA, Shoham Y, Steitz TA (1996) Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose. EMBO J 15(21):5739–5751

    CAS  Google Scholar 

  • Zhao Z, Lu W, Dun B, Jin D, Ping S, Zhang W, Chen M, Xu MQ, Lin M (2008) Purification of green fluorescent protein using a two-intein system. Appl Microbiol Biot 77(5):1175–1180. doi:10.1007/s00253-007-1233-0

    Article  CAS  Google Scholar 

  • Zhou X, Cai S, Hong A, You Q, Yu P, Sheng N, Srivannavit O, Muranjan S, Rouillard JM, Xia Y, Zhang X, Xiang Q, Ganesh R, Zhu Q, Matejko A, Gulari E, Gao X (2004) Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences. Nucleic Acids Res 32(18):5409–5417. doi:10.1093/nar/gkh879

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant-in-aid from the Anhui Provincial Natural Science Foundation (grant no. 090413075), the State Education Ministry and Specialized Research Fund for the Doctoral Program of Higher Education of China (grant no. 20093402120027), the National Basic Research Program of China (grant no. 2011CBA00801), and the Fundamental Research Funds for the Central Universities (grant no. WK2070000007). The authors declare that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. School of Life Science, University of Science and Technology of China, Hefei, Anhui, People’s Republic of China

    Wen Wan, Dongmei Wang, Xiaolian Gao & Jiong Hong

  2. Hefei National Laboratory for Physical Science at the Microscale, Hefei, Anhui, People’s Republic of China

    Wen Wan, Xiaolian Gao & Jiong Hong

  3. Department of Biology and Biochemistry, University of Houston, Houston, TX, USA

    Xiaolian Gao

Authors
  1. Wen Wan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dongmei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiaolian Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jiong Hong
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jiong Hong.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 49 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wan, W., Wang, D., Gao, X. et al. Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast. Appl Microbiol Biotechnol 91, 789–798 (2011). https://doi.org/10.1007/s00253-011-3373-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-011-3373-5

Keywords

Search

Navigation