Enzymic preparation of protein G-peroxidase conjugates catalysed by transglutaminase.

Abstract:

:Transglutaminases (TGases, EC 2.3.2.13) have proved to be valuable enzymes for site-directed protein coupling via N(epsilon)-(gamma-L-glutamyl)lysine bonds. Their use in conjugate synthesis would overcome many problems caused by chemical reagents. In this approach, we show for the first time that two proteins with different functionalities, namely soybean peroxidase and protein G, can be cross-linked by bacterial TGase with retention of their activities. Soybean peroxidase and protein G were chosen for the enzymic preparation of a bifunctional conjugate among a series of other TGase substrates detected by enzymic incorporation of small fluorescent or biotinylated molecules. The highest yields of conjugate were obtained with a 15-fold excess of peroxidase in phosphate buffer, pH 7.0. Size exclusion chromatography enabled both purification of the conjugates and recovery of the starting materials. Analysis of bifunctionality revealed the coupling of protein G with an average of three peroxidase molecules.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Bechtold U,Otterbach JT,Pasternack R,Fuchsbauer HL

doi

10.1093/oxfordjournals.jbchem.a022600

keywords:

subject

Has Abstract

pub_date

2000-02-01 00:00:00

pages

239-45

issue

2

eissn

0021-924X

issn

1756-2651

journal_volume

127

pub_type

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