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High level expression of peptides and proteins using cytochrome $b_5$ as a fusion host

Mitra, Ashima and Chakrabarti, Kalyan Sundar and Hameed, Shahul MS and Srinivas, Kalyan V and Kumar, Ganesan Senthil and Sarma, Siddhartha P (2005) High level expression of peptides and proteins using cytochrome $b_5$ as a fusion host. In: Protein Expression and Purification, 41 (1). pp. 84-97.

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A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome $b_5$ has been designed. The ability of cytochrome $b_5$ to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., $\alpha$ hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, $Na_V1.2$. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel puriWcation protocol has been designed to purify the fusion proteins using metal aYnity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome $b_5$ based fusion system for the production of peptides and small proteins (<15 kDa).

Item Type: Journal Article
Additional Information: The copyright of this article belongs to Elsevier.
Keywords: Fusion proteins;Cytochrome b5;Protein solubility;Protein expression;Metal aYnity chromatography;Histidine tagged fusion proteins
Department/Centre: Division of Biological Sciences > Molecular Biophysics Unit
Depositing User: Nagaraj Vaidya
Date Deposited: 27 Feb 2006
Last Modified: 19 Sep 2010 04:24
URI: http://eprints.iisc.ac.in/id/eprint/5632

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