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Updated: May 11, 2026
The Multifaceted Benefits of Protein Co-expression in Escherichia coli
Published on: February 5, 2015
Strategic truncation improves recombinant expression of buffalo Fibulin-5 (FBLN5) in Escherichia coli
Deviyani Mahajan1, Neha Sarova2, Jyoti Rustagi2
1Department of Biotechnology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, 144411, Punjab, India.
Background:
Accurate early pregnancy detection is crucial for enhancing herd productivity in bovines. There is growing interest in identifying reliable protein biomarkers that can be used to develop rapid, sensitive, and cost-effective diagnostic tools. This study focused on buffalo fibulin-5 (FBLN5), an extracellular matrix glycoprotein involved in tissue remodeling and cell adhesion, and a potential early pregnancy biomarker.
Methods:
In this study, we used the pET-32a(+) expression vector and three E. coli expression hosts. Heat-shock transformation was performed in chemically competent cells (CaCl2 method), and the expression was optimized at 18 °C and 37 °C. Recombinant truncated FBLN5 was purified through immobilized metal affinity chromatography (IMAC) on nickel resin and subsequently characterized by SDS-PAGE and Western blotting.
Results:
Optimization of various expression conditions, such as temperature, induction, incubation time, and additives, did not result in any detectable expression of full-length FBLN5. Therefore, a truncated version of FBLN5 was designed based on the predicted antigenic regions to increase its immunogenicity and simplify its recombinant expression. Truncated FBLN5 (Trx-tagged and 6 × His-tagged) expressed well in BL21Codon Plus (DE3)-RIL and Lemo21(DE3) at 18 °C with 1 mM IPTG induction. Furthermore, we successfully purified soluble truncated FBLN5 and confirmed its molecular weight (∼36 kDa) and identity using western blotting with enhanced chemiluminescence (ECL).
Conclusion:
Truncation of full-length FBLN5 resulted in successful cloning, expression, and purification of this otherwise difficult to express protein. This study provides a solid foundation for future immunological studies aimed to evaluate FBLN5 potential as diagnostic marker in early pregnancy detection in bovine species.
