Influence of Tags on the Binding Affinity of Acyl-CoA Binding Protein

Jizhong Han,Mengmeng Liu,Yu Chen,Yunlong Sun,Haoran Li,Bin Zeng

doi:10.3844/ajbbsp.2019.13.22

Abstract

Tagged fusion proteins are frequently employed for protein purification methods, but their effects on protein function and binding affinity are rarely studied. Here we expressed recombinant protein Acyl-CoA Binding Protein (ACBP) cloned from the full-length cDNA of Aspergillus oryzae and Saccharomyces cerevisiae. ACBP was expressed in Escherichia coli fused to a Maltose-Binding Protein (MBP) and Histidine-tag fusion. Recombinant ACBP was purified using affinity chromatography columns and high protein purity was achieved. Microscale Thermophoresis (MST) binding assays showed that recombinant AoAcbp1 had a greater affinity for Palmitoyl-CoA (Kd = 35 nM) and Stearoyl-CoA (Kd = 23 nM) whilst recombinant ScAcbp had a greater affinity for Myristoyl-CoA (Kd = 31 nM) and Palmitoyl-CoA (Kd = 51 nM). In addition, MBP tagged ACBP had comparable binding affinities to His-tagged ACBP. Taken together, these data highlight that the size of the tagged fusion protein does not influence protein ACBP binding affinity.

Highlights

Aspergillus oryzae and Saccharomyces cerevisiae are frequently used in industrial biotechnology (Liu et al, 2014) for fatty acid biosynthesis and metabolism, in the fermented food industry (Piras, 2014)
In S. cerevisiae, acylCoA Binding Protein (ACBP) (ScAcbp) is a highly conserved 10 kDa protein that is required for vacuole function and ceramide synthesis (Faergeman et al, 2004), ScAcbp activity reduces the levels of hydroxy-C26:0 fatty acids, influences sphingolipid synthesis and regulates the expression of genes involved in fatty acid desaturation (Gaigg et al, 2001)
In past studies, when Maltose-Binding Protein (MBP) was used as fusion protein, whether it would affect the function of ACBP, it was uncertainty

Summary

Introduction

Aspergillus oryzae and Saccharomyces cerevisiae are frequently used in industrial biotechnology (Liu et al, 2014) for fatty acid biosynthesis and metabolism, in the fermented food industry (Piras, 2014). Fatty acid oxidation is mediated by fatty acyl-CoA and acylCoA Binding Protein (ACBP) (Wei et al, 2007). Previous studies have characterized the functions of ACBP in A. oryzae and S. cerevisiae. In S. cerevisiae, ACBP (ScAcbp) is a highly conserved 10 kDa protein that is required for vacuole function and ceramide synthesis (Faergeman et al, 2004), ScAcbp activity reduces the levels of hydroxy-C26:0 fatty acids, influences sphingolipid synthesis and regulates the expression of genes involved in fatty acid desaturation (Gaigg et al, 2001). In past studies, when MBP was used as fusion protein, whether it would affect the function of ACBP, it was uncertainty. MBP-ACBP and His-tagged ACBP were produced to high purity for the assessment of their binding affinities using Microscale Thermophoresis (MST) technology. We reproducibly performed experimental binding measurements using minimal amounts of protein and fragments (Gudim et al, 2017)

Materials and Methods

Results and Discussion

Funding Information