Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection.

نویسندگان

  • Fujie Tanaka
  • Roberta Fuller
  • Hyunbo Shim
  • Richard A Lerner
  • Carlos F Barbas
چکیده

Aldolase antibodies that operate via an enamine mechanism were developed by in vitro selection. Antibody Fab phage display libraries were created where the catalytic active site residues of aldolase antibodies 38C2 and 33F12 were combined with a naive human antibody V gene repertoire. Selection from these libraries with 1,3-diketones covalently trapped the amino groups of reactive lysine residues by formation of stable enaminones. The selected aldolase antibodies retained the essential catalytic lysine residue and its function in altered and humanized primary antibody structures. The substrate specificity of the aldolase antibodies was directly related to the structure of the diketone used for selection. The k(cat) values of the antibody-catalyzed retro-aldol reactions were correlated with the K(d) values, i.e. the reactivities of the selected aldolase antibodies for the corresponding diketones. Antibodies that bound to the diketone with a lower K(d) value displayed a higher k(cat) value in the retro-aldol reaction, and a linear relationship was observed in the plots of logk(cat) versus logK(d). These results indicate that selections with diketones directed the evolution of aldolase antibodies in vitro that operate via an enamine mechanism. This strategy provides a route to tailor-made aldol catalysts with different substrate specificities.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Synthesis of five enantiomerically pure haptens designed for in vitro evolution of antibodies with peptidase activity.

A series of five haptens have been synthesized for use in vitro selection experiments from combinatorial antibody libraries. Haptens were designed for the recruitment of serine and cysteine protease reaction mechanisms for the cleavage of Phe-Ala and Phe-Phe (L,L) dipeptide analogues. For the selection of transition state stabilization, PheP(O)Ala (7) and PheP(O)Phe (10) derivatives were synthe...

متن کامل

Hydrogen evolution from catalytic hydrolysis of NaBH4: Comparative study between the catalytic activity of TiO2 nanotubes with various arrangements

Nowadays, a lot of efforts have been applied to find an appropriate catalyst for generating hydrogen from NaBH4. Hence in the current study, various nanostructures of TiO2 were employed to obtain an insight into how the different support catalysts effect on the hydrolysis rate of NaBH4. For this aim, disordered filaments (DF-NTs) and ordered free-standing TiO2 nanotubes (FS-NTs) were fabricated...

متن کامل

آپتامرها و کاربردهای بیولوژیکی-درمانی آنها

Aptamers are the artificial single-stranded DNA or RNA sequences (more recently, peptides) that fold into secondary and tertiary structures making them bind to certain targets with extremely high specificity. Aptamers were reported for the first time in 1990, a number of their unique features make them a more effective choice than antibodies. Aptamers typically generated through Systematic Ev...

متن کامل

Origin of Catalytic Activity Differences between Phosphine and Phosphine Oxide-Based Structures in the Water-Crosslinkable Polyalkoxysilane Composition

Organocatalysts have attracted enormous interest in the water-crosslinking reaction in silane-grafted polyolefins (SGPOs) system owing to their simplicity, low toxicity and environmentally benign nature compared to organotin catalysts, which are most used in SGPOs system. We focus on organophosphorus compounds including four structure types as organocatalysts; phosphoric acids, phosphoric ester...

متن کامل

The effect of antisperm antibodies measured by direct mixed agglutination reaction on in vitro fertilization rate

Background: Although in vitro fertilization (IVF) has been found to bypass the inhibitory effect of high levels of antisperm antibodies on sperm motility, fertilization rates are reduced in approximately 40% of the attempts. In this study, we investigated the relationship between antisperm antibodies measured by the direct mixed agglutination reaction (MAR) and the fertilization rate in inferti...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Journal of molecular biology

دوره 335 4  شماره 

صفحات  -

تاریخ انتشار 2004