Expression of functional human retinol-binding protein in Escherichia coli using a secretion vector

Area of Science:

• Molecular Biology
• Protein Expression
• Biochemistry

Background:

• Human serum retinol-binding protein (sRBP) is crucial for vitamin A transport.
• Efficient expression of structurally native sRBP in a heterologous system like E. coli is challenging.
• Understanding sRBP-ligand and sRBP-receptor interactions is vital for biological studies.

Purpose of the Study:

• To develop a method for expressing functional human serum retinol-binding protein (sRBP) in Escherichia coli.
• To produce recombinant sRBP (rRBP) that is structurally indistinguishable from native sRBP.
• To characterize the molecular recognition properties of the expressed rRBP.

Main Methods:

• Constructed secretion vectors by fusing RBP cDNA with the OmpA signal sequence.
• Subcloned DNA fragments into expression plasmids (pKS-Bluescript) with T7 promoter.
• Optimized expression by deleting the 3' untranslated region of RBP cDNA.
• Purified secreted RBP using transthyretin-affinity chromatography after retinol charging.
• Assessed binding affinities for retinol, transthyretin, and the cell-surface receptor.

Main Results:

• Initial constructs yielded no detectable RBP expression in E. coli.
• Subcloning and optimization (pOmp-RBP2, pOmp-RBP3) led to detectable periplasmic secretion of RBP, with a 20-fold improvement after 3' UTR deletion.
• Purified rRBP demonstrated characteristic molecular recognition properties, binding retinol, transthyretin, and its receptor.
• rRBP exhibited similar retinol-binding affinity to native sRBP but a 6-8 fold higher Bmax for receptor binding.

Conclusions:

• Successfully engineered E. coli to express functional human serum retinol-binding protein (sRBP).
• The optimized expression system yields rRBP with native-like binding affinities but altered receptor interaction dynamics.
• A significant portion of native sRBP in serum may be functionally compromised in receptor interaction.