Expression, purification and applications of staphylococcal Protein A fused to cellulose-binding domain

Etai Shpigel, Arie Goldlust, Adi Eshel, Idit Kaplan Ber, Gilat Efronif, Yossi Singer, Ilan Levy, Mara Dekel, Oded Shoseyov*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

44 Scopus citations


Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics. Immobilization is required for most of these applications, Here we describe a genetic-engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose-binding domain (CBD) derived from the cellulose-binding Protein A of Clostridium cellulovorans. The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH, ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgC from different sources was determined. The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgC at a ratio of 1 mol of ProtA-CBD/2 mol of human IgC, and can purify up to 11.6 mg/ml rabbit IgC from a serum. The ability to bind functionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated. Our results indicate that a combination of CBD-affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non-specific binding; and (ii) CBD-fusion proteins bind directly to cellulose at high density. A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a Western blot.

Original languageAmerican English
Pages (from-to)197-203
Number of pages7
JournalBiotechnology and Applied Biochemistry
Issue number3
StatePublished - 2000


  • Affinity chromatography
  • IgG purification
  • Immobilization


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