Functional expression and purification of the untagged C-terminal domain of MMP-2 from Escherichia coli inclusion bodies

2020 ◽  
Vol 176 ◽  
pp. 105726
Author(s):  
Yi Zhou ◽  
Chunmao He
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Functional Expression ◽  
Expression And Purification ◽  
Terminal Domain

scholarly journals Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli

2004 ◽  
Vol 70 (12) ◽  
pp. 6968-6976 ◽  
Author(s):  
Taek Ho Yang ◽  
Jae Gu Pan ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Cell Viability ◽  
Pseudomonas Putida ◽  
Outer Membrane ◽  
Fusion Proteins ◽  
Functional Expression ◽  
E Coli ◽  
Terminal Domain ◽  
Anchoring Motif ◽  
Periplasmic Enzyme

ABSTRACT The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme β-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.

scholarly journals Optimization of the Protocol for the Isolation and Refolding of the Extracellular Domain of HER2 Expressed in Escherichia coli

Acta Naturae ◽  
2014 ◽  
Vol 6 (2) ◽  
pp. 106-109 ◽  
Author(s):  
V. V. Dolgikh ◽  
I. V. Senderskiy ◽  
G. V. Tetz ◽  
V. V. Tetz
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Extracellular Domain ◽  
Eukaryotic Expression ◽  
Soluble Form ◽  
Expression And Purification ◽  
Recombinant Product ◽  
Oncological Diseases ◽  
Epidermal Growth ◽  
Oncogene Protein

Receptor 2 of the human epidermal growth factor (HER2/neu, c-erbB2) is a 185 kDa proto-oncogene protein characterized by an overexpression in some oncological diseases, including 30% of mammary glands cancers, as well as tumors in the ovary, stomach and other organs of the human body. Since HER2- tumor status testing is the essential part of a successful cancer treatment, the expression and purification of substantial amounts of the extracellular domain (ECD) of HER2 is an important task. The production of ECD HER2 in Escherichia coli has several advantages over the use of eukaryotic expression systems, but the bulk of the recombinant product in bacteria accumulates as insoluble protein inclusion bodies. In this study, we obtained ECD HER2 in Escherichia coli as insoluble inclusion bodies and elaborated a simple, efficient, and fast protocol for the solubilization, refolding, and isolation of the protein in soluble form.

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