Isolation of a new human scFv antibody recognizing a cell surface binding site to CEACAM1. Large yield production, purification and characterization in E. coli expression system

Abstract Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis. Key points • First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS • CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays • CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice. Graphical abstract

Download Full-text

Conserved regulation of mesenchymal gene expression by Fgf-8 in face and limb development

Development ◽

10.1242/dev.126.2.221 ◽

1999 ◽

Vol 126 (2) ◽

pp. 221-228 ◽

Cited By ~ 1

Author(s):

A.S. Tucker ◽

A. Al Khamis ◽

C.A. Ferguson ◽

I. Bach ◽

M.G. Rosenfeld ◽

...

Keyword(s):

Gene Expression ◽

Limb Development ◽

Regulation Of Gene Expression ◽

Limb Bud ◽

Lim Domain ◽

Surface Binding ◽

Mandibular Arch ◽

Cell Surface Binding ◽

Signalling Molecule ◽

A Cell

Clim-2 (NLI, Lbd1) is one of two related mouse proteins that interact with Lim-domain homeoproteins. In the mouse, embryonic expression of Clim-2 is particularly pronounced in facial ectomesenchyme and limb bud mesenchyme in association with Lim genes, Lhx-6 and Lmx-1 respectively. We show that in common with both these Lim genes, Clim-2 expression is regulated by signals from overlying epithelium. In both the developing face and the limb buds we identify Fgf-8 as the likely candidate signalling molecule that regulates Clim-2 expression. We show that in the mandibular arch, as in the limb, Fgf-8 functions in combination with CD44, a cell surface binding protein, and that blocking CD44 binding results in inhibition of Fgf8-induced expression of Clim-2 and Lhx-6. Regulation of gene expression by Fgf8 in association with CD44 is thus conserved between limb and mandibular arch development.

Download Full-text

Interaction of Poliovirus with its Cell Surface Binding Site

Virology ◽

10.1006/viro.1994.1270 ◽

1994 ◽

Vol 201 (1) ◽

pp. 107-115 ◽

Cited By ~ 12

Author(s):

James A. Bibb ◽

Gary Witherell ◽

Günter Bernhardt ◽

Eckard Wimmer

Keyword(s):

Cell Surface ◽

Binding Site ◽

Surface Binding ◽

Cell Surface Binding ◽

Surface Binding Site

Download Full-text

Construction of an Artificial Cell Capable of Protein Expression at Low Temperatures Using a Cell Extract Derived from Pseudomonas fluorescens

Processes ◽

10.3390/pr9020212 ◽

2021 ◽

Vol 9 (2) ◽

pp. 212

Author(s):

Mana Fukumoto ◽

Taishi Tonooka

Keyword(s):

Protein Expression ◽

Pseudomonas Fluorescens ◽

Fluorescent Proteins ◽

Expression System ◽

Cell Extract ◽

Bilayer Membrane ◽

E Coli ◽

Cold Environments ◽

Artificial Cell ◽

A Cell

A liposome-based artificial cell (LBAC) consists of a liposome encapsulating a cell-free protein expression system (CFPES) and protein-encoding DNA. It is surrounded by a lipid bilayer membrane and synthesizes proteins that resemble actual cells. Hence, they have been one of the most studied artificial cells. According to recent studies, they have been able to sense bio-functional molecules by synthesizing fluorescent proteins in response to target molecules. Therefore, they are expected to be used as biosensors. However, previously reported LBACs encapsulated the CFPES derived from Escherichia coli, resulting in the most productive protein expression at 20–40 °C. To broaden the range of their working temperatures to lower temperatures, in this study, we constructed LBACs using a CFPES derived from Pseudomonas fluorescens that grows at a temperature range of 4 °C to 30 °C. We then demonstrated that the constructed LBAC expressed proteins at 8 °C and that, the protein expression capability of the LBAC derived from P. fluorescens was four-fold higher than that derived from E. coli at 8 °C. This study will pave the way for the development of artificial cell-based biosensors that work in cold environments or for the synthesis of heat-labile proteins in LBACs.

Download Full-text