Enhanced stabilization of a stable single domain antibody for SEB toxin by random mutagenesis and stringent selection.

نویسندگان

  • Kendrick B Turner
  • Dan Zabetakis
  • Ellen R Goldman
  • George P Anderson
چکیده

Single domain antibodies, recombinant variable heavy domains derived from the unique heavy-chain only antibodies found in camelids and sharks, are exceptionally rugged due to their ability to refold following heat or chemical denaturation. In addition, a number of single domain antibodies have been found to possess high melting points which provide an even greater degree of stability; one of these, llama-derived A3, is a binder of Staphylococcal enterotoxin B and has a Tm of 83.5 °C. In this work, we utilized random mutagenesis and stringent selection in an effort to obtain variants of A3 with even higher melting points. This effort resulted in the selection of a double mutant, A3-T28I-S72I, which has a melting point of 90.0 °C and near wild-type affinity for the target antigen. We further characterized the mutations individually to determine that while both contributed to the thermal stabilization, the T28I mutation accounted for ∼ 4.1 °C of the 6.5 °C increase. This work demonstrates that by the addition of relatively subtle changes it is possible to further improve the melting temperature of single domain antibodies that are already remarkably stable.

برای دانلود رایگان 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

Isolation of a Highly Thermal Stable Lama Single Domain Antibody Specific for Staphylococcus aureus Enterotoxin B

BACKGROUND Camelids and sharks possess a unique subclass of antibodies comprised of only heavy chains. The antigen binding fragments of these unique antibodies can be cloned and expressed as single domain antibodies (sdAbs). The ability of these small antigen-binding molecules to refold after heating to achieve their original structure, as well as their diminutive size, makes them attractive ca...

متن کامل

Mutagenesis of Xanthomonas campestris and Selection of Strains with Enhanced Xanthan Production

Xanthan gum is a microbial polysaccharide of great commercial importance as it has unusual rheological properties in solution and consequent range of applications. In this study, a series of mutants were isolated from Xanthomonas campestris PTCC 1473 by ethyl methanesulfonate mutagenesis. The polysaccharide yield of one mutant, XC1473E2, was 30% better than that of the parent strain. It also sh...

متن کامل

Construction and Expression of Hepatitis B Surface Antigen Escape Variants within the "a" Determinant by Site Directed Mutagenesis

Background: The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. Objectives: To construct clinically relevant recombinant muta...

متن کامل

Construction of a single-chain variable-fragment antibody against the superantigen Staphylococcal enterotoxin B.

Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detect...

متن کامل

Serratia marcescens B4A chitinase thermostability enhancement by S390I QuikChange site directed mutagenesis

Thermostable chitinases are useful for industrial and biotechnological applications. This paper reports the stabilization of chitinase from Serratia marcescens B4A through rational mutagenesis. Changing of Ser 390 to Ile in S. marcescens. The stabilization was enhanced through entropic stabilization by reduction of the loop length and also by increasing of the beta chain length. With this repla...

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

عنوان ژورنال:
  • Protein engineering, design & selection : PEDS

دوره 27 3  شماره 

صفحات  -

تاریخ انتشار 2014