S108 Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S96–S113

to discover novel enzyme Sortase-LPW that can catalyze proteinmodification with functional molecule.

doi:10.1016/j.jbiosc.2009.08.314

EP-P27

Site-specific streptavidin modification using sortase

Takuya Matsumoto,1 Shiori Sawamoto,1 Takayuki Sakamoto,1 TsutomuTanaka,2 and Akihiko Kondo1

Department of Chemical Science and Engineering, Graduate School ofEngineering, Kobe University, Kobe, Japan1 and Organization ofAdvanced Science and Technology, Kobe University, Kobe, Japan2

Streptavidin has tight and specific biotin-binding affinity. Strepta-vidin modified with other protein or small molecule is widely used forbiomolecule labeling, purification and immobilization using biotin-binding affinity. Usually, modified streptavidin is prepared by chemicalmodification. However, biotin-binding affinity of modified streptavi-din prepared by chemical modification is not always equivalent ofnative streptavidin. Then we proposed site-specific modification ofstreptavidin using enzyme, to expand its application more widely. Weuse sortaseA, transpeptidase from Staphylococcus aureus as proteinmodification enzyme. SortaseA selectively recognizes C-terminalLPXTG motif, and cleaves between threonine and glycine, and joinscarboxyl group of threonine to amino group of GGGGG motif.Streptavidin which tagged LPETG motif that is recognition sequenceof sortaseA was expressed in Escherichia coli. Because expressedstreptavidin formed inclusion bodies, inclusion bodies were refoldedby dialysis. After refolding, biotin-binding affinity of tetramericstreptavidin was retained as same as wild-type streptavidin. Weconjugated GGGGG-tagged EGFP to LPETG-tagged streptavidin usingsortaseA. As a result of SDS-PAGE, streptavidin was site-specificallyconjugated GFP. These results show thatwe successfully demonstratedsite-specific modification of streptavidin using sortaseA, and we alsosuccessfully conjugated other proteins, such as ZZ-domain and glucoseoxidase to streptavidin using sortaseA. Our strategy provides apowerful tool for site-specific conjugation with streptavidin.

doi:10.1016/j.jbiosc.2009.08.315

EP-P28

Functional analysis of mutant human somatostatin receptor usinga yeast-based fluorescence reporter assay

Shota Togawa,1 Jun Ishii,2 Tsutomu Tanaka,2 and Akihiko Kondo1

Department of Chemical Science and Engineering, Graduate School ofEngineering, Kobe University, Kobe, Japan1 and Organization ofAdvanced Science and Technology, Kobe University, Kobe, Japan2

A large number of hormones, neurotransmitters and sensorystimuli exert their effects on cells and organisms by binding toGPCRs (G-protein coupled receptors). Heterotrimeric G-proteinstransduce the binding of ligands to these receptors into intracellularsignals, which underlie many physiological responses of tissues and

organisms such as increased blood pressure and vasoconstriction. Inorder for a GPCR to elicit intracellular signalling, it must first gothrough a series of biosynthetic events aimed at sending theappropriate quantity of properly folded functional receptors to theplasma membrane. Glycosylation is a common post-translationalmodification in the GPCR family. Classically, glycosylation has beenthought to regulate cell-surface expression of receptors. However,more recently, various studies have pointed towards a role forglycosylation in direct regulation of receptor function.

In this study, we examine a role of putative N-linked glycosyla-tion sites on somatostatin receptor subtype 5 (SSTR5) by anengineered yeast Saccharomyces cerevisiae which expressesenhanced green fluorescent protein (EGFP) in response to the signaltransduction. Single mutation of the N-glycosylation site N13A orN26A had a small effect on signal transduction. The double mutantN13A/N26A lacking both glycosylation sites showed a significantreduction in expression of EGFP. To evaluate the plasma membranelocalization of mutated GPCR, these mutants fusing with EGFP at thecarboxy terminus were analyzed by confocal laser scanningmicroscope. As a result, mutation of the N-glycosylation sitesexerted no influence on cell-surface expression of this receptor.These results suggested that N-glycosylation on SSTR5 plays animportant role in the physiological function.

doi:10.1016/j.jbiosc.2009.08.316

EP-P29

Enzyme-mediated antibody-protein conjugation

Takayuki Sakamoto,1 Tsutomu Tanaka,2 and Akihiko Kondo1

Department of Chemical Science and Engineering, Graduate School ofEngineering, Kobe University, Kobe, Japan1 and Organization ofAdvanced Science and Technology, Kobe University, Kobe, Japan2

Antibody has molecular recognition functions and antibody-enzyme (and some kinds of proteins) fusion protein has been usedin various kind of field. For example, the fusion protein has been feltuseful in diagnosis of a disease and determination environmentalpollutant. However, current method to produce fusion proteins hasseveral problems such as loss of function and heterogeneity of theproducts, because of random reaction. In this study, we developed theenzyme-based strategy to conjugate antibody and functional proteins.We can control site specific reaction of protein by the use of enzyme.We have focused on SortaseA from Staphylococcus aureus astranspeptidase. SortaseA recognize the sequence of amino acids“LPETG” and cleaves between T and G. Then, it connects proteinwith pentaglycine of its N-terminus. First, the substrate sequences aregenetically introduced to the antibodies and functional proteins. Thenthe expressed tagged proteins are conjugated by addition of SortaseA.However, it is difficult to carry out genetic manipulation againstantibody. Therefore, we focused on ZZ domain that is easy tomanipulate its gene and has affinity for antibody. Using the taggedZZ domain, we successfully conjugate antibody and functionalproteins such as enzymes. In this study, we constructed ZZ-LPETGand GGGGG-Luciferase. So by mixing ZZ-LPETG and GGGGG-Lucifer-ase and SortaseA, we got ZZ-Luciferase conjugate. As a result, we canproduce antibody–protein conjugate without loss of its function andheterogeneity of the products.

doi:10.1016/j.jbiosc.2009.08.317