Comparison of the efficiencies of different affinity tags in the purification of a recombinant secretory protein expressed in silkworm larval hemolymph

• Published in: Biotechnology and bioprocess engineering Vol. 14; no. 3; pp. 281 - 287
• Main Authors: Dojima, Takashi (Shizuoka University, Shizuoka, Japan), Nishina, Takuya (Shizuoka University, Shizuoka, Japan), Kato, Tatsuya (Shizuoka University, Shizuoka, Japan), Ueda, Hiroshi (University of Tokyo, Tokyo, Japan), Park, Enoch Y. (Shizuoka University, Shizuoka, Japan), E-mail: yspark@agr.shizuoka.ac.jp
• Format: Journal Article
• Language: English
• Published: Heidelberg The Korean Society for Biotechnology and Bioengineering 01.06.2009; Springer Nature B.V; 한국생물공학회
• Subjects: affinity tag; Antibodies; antibody; Bioreactors; Biotechnology; BOMBYX MORI; Bombyx mori NPV; Chemistry; Chemistry and Materials Science; Cloning; Comparative analysis; Contaminants; E coli; Efficiency; Gene amplification; Industrial and Production Engineering; Larvae; Processes; Protein expression; Proteins; silkworm larvae; 생물공학; Bombyx mori
• ISSN: 1226-8372; 1976-3816
• DOI: 10.1007/s12257-008-0258-2

Silkworms are useful bioreactors for heterologous protein expression when used in conjunction with the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. However, purification from silkworm hemolymph is difficult since it contains various kinds of proteins. In this study, we investigated an effective single-step method for the purification of affinity-tagged single-chain antibody variable region fragment (scFv) from silkworm larval hemolymph. A 5-fold higher expression level was obtained when scFv was fused with the His tag than when it was fused with the Strep Ⅱ or GST tags. However, the His tag was inadequate for single-step purification since it led to the nonspecific binding of contaminants. The purification recoveries of GST-, Strep Ⅱ-, and His-tagged scFvs were 91.8%, 43.7%, and 27.2%, respectively. The specific amount of single-step purified GST-tagged scFv was 2.2~2.7 fold higher than the amounts of the His- and Strep Ⅱ-tagged constructs. The purities of Strep Ⅱ- and GST-tagged scFvs in the eluent were 98.4% and 83.0%, respectively. Thus, both the short peptide Strep Ⅱ and GST protein are suitable fusion tags for the affinity purification of proteins from silkworm larvae.