Title

Use of the amicyanin signal sequence for efficient periplasmic expression in E-coli of a human antibody light chain variable domain

Authors

Authors

B. A. Dow; S. A. Tatulian;V. L. Davidson

Comments

Authors: contact us about adding a copy of your work at STARS@ucf.edu

Abbreviated Journal Title

Protein Expr. Purif.

Keywords

Signal sequence; Amicyanin; Protein folding; Protein expression; Antibody; INCLUSION-BODIES; PARACOCCUS-DENITRIFICANS; PROTEIN; AGGREGATION; PURIFICATION; FRAGMENTS; GENE; Biochemical Research Methods; Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology

Abstract

Periplasmic localization of recombinant proteins offers advantages over cytoplasmic protein expression. In this study signal sequence of amicyanin, which is encoded by the mauC gene of Paracoccus denitrificans, was used to express the light chain variable domain of the human kappa IO8/O18 germline antibody in the periplasm of Escherichia coli. The expressed protein was purified in good yield (70 mg/L of culture) in one step from the periplasmic fraction by affinity chromatography using an engineered hexahistidine tag. Circular dichroism spectroscopy was used to determine if the secondary and tertiary structures of the protein and its thermal stability corresponded to those of the native folded protein. The expressed and purified protein was indeed properly folded and exhibited a reasonable thermal transition temperature of 53 degrees C. These results indicate that the amicyanin signal sequence may be particularly useful for prokaryotic expression of proteins which are prone to mis-folding, aggregation or formation of inclusion bodies, all of which were circumvented in this study. (C) 2014 Elsevier Inc. All rights reserved.

Journal Title

Protein Expression and Purification

Volume

108

Publication Date

1-1-2015

Document Type

Article

Language

English

First Page

9

Last Page

12

WOS Identifier

WOS:000352176100002

ISSN

1046-5928

Share

COinS