Site-Specific, Enzymatic Biotinylation of Recombinant Proteins in Spodoptera frugiperda Cells Using Biotin Acceptor Peptides 1 Siobain Duffy,* Kwei-Lan Tsao,† and David S. Waugh* ,2 *ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702-1201; and †Roche Research Center, Hoffmann La-Roche, Inc., Kingsland Street, Nutley, New Jersey 07110 Received January 28, 1998 Site-specific, enzymatic biotinylation of recombi- nant proteins can be exploited to circumvent many problems associated with the use of biotinylating re- agents in vitro and to overcome some of their inherent limitations. Additionally, biotinyl proteins can be pu- rified to near-homogeneity in a single step under na- tive conditions. Here we report that a biotin acceptor peptide (BAP) substrate for Escherichia coli biotin ho- loenzyme synthetase (BirA) can be used to label re- combinant proteins with biotin in Spodoptera frugi- perda (Sf9) cells, and we describe a collection of baculovirus transfer vectors specifically designed for this purpose. These BioBac vectors will greatly ex- pand the range of proteins to which this technology can be applied. © 1998 Academic Press Key Words: enzymatic biotinylation; baculovirus; bi- otin acceptor peptide; BirA. Biotin (vitamin H) is frequently conjugated to pro- teins so that they can be immobilized on avidin- or streptavidin-coated surfaces for various kinds of li- gand-binding experiments. A multitude of chemical re- agents have been developed for this purpose (1). How- ever, although each reagent is specific for a certain type of functional group, none is inherently site selec- tive. For example, a reagent that only reacts with pri- mary amino groups can potentially modify any of the lysine side chains in a protein, which are likely to be scattered over its surface. Biotinylation at multiple sites increases the potential for direct and indirect inactivation of the protein by the biotinylating reagent. Direct inactivation can occur when some of the reactive groups (or the neighboring atoms) are crucial for the biological activity of the protein. Although this problem can sometimes be overcome by choosing a different reagent, regardless of which reagent is used, some derivatized proteins are likely to be inactivated indi- rectly due to immobilization in a sterically unfavorable orientation. Additionally, because biotinylation with chemical reagents typically yields a heterogeneous mixture of reaction products, ensuring batch-to-batch reproducibility can be a formidable problem. All cells contain a small number of proteins to which biotin is covalently attached as a prosthetic group (2, 3). Biotin carboxyl carrier protein (BCCP), 3 a subunit of acetyl-CoA carboxylase, is the only biotinyl protein in Escherichia coli. Biotin is coupled via an amide linkage to the -amino group of Lys122 in BCCP by the enzyme biotin holoenzyme synthetase (BirA) in an ATP-dependent reaction (4, 5). Recombinant proteins can be labeled with biotin at a single, predetermined site, albeit indirectly, by producing them as BCCP fu- sion proteins in E. coli (6 –10). This approach circum- vents several problems with chemical reagents. First, enzymatic biotinylation at a specific site ensures that all molecules will be immobilized in the same (bioac- tive) orientation, thereby resulting in the highest pos- sible specific activity. Additionally, since the chemistry is performed in vivo, there are no hazardous reagents to handle and dispose of. Finally, the specificity of the 1 By acceptance of this article, the publisher or recipient acknowl- edges the right of the U.S. Government and its agents and contrac- tors to retain a nonexclusive royalty-free license in and to any copy- right covering the article. 2 To whom correspondence should be addressed. Fax: (301) 846- 7148. E-mail: waughd@ncifcrf.gov. 3 Abbreviations used: BCCP, biotin carboxyl carrier protein; BirA, biotin holoenzyme synthetase; BAP, biotin acceptor peptide; TBS, Tris-buffered saline. 122 0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ANALYTICAL BIOCHEMISTRY 262, 122–128 (1998) ARTICLE NO. AB982770