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

Corresponding Author: Bin Zeng Jiangxi Key Laboratory of Bioprocess Engineering and Co-Innovation Center for Invitro Diagnostic Reagents and Devices of Jiangxi Province, College of Life Sciences, Jiangxi Science and Technology Normal University, Nanchang 330013, China Email: zengtx001@aliyun.com 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 AcylCoA 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 Histidinetag 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.


Introduction
Aspergillus oryzae and Saccharomyces cerevisiae are frequently used in industrial biotechnology (Liu et al., 2014) for fatty acid biosynthesis and metabolism, particularly in the fermented food industry (Piras, 2014). A. oryzae is used in the fermentation of rice-based food, soy and sake (Knuf and Nielsen, 2012), due to its high levels of protein secretion and ability to perform posttranslational modifications (Meyer et al., 2011). In addition, S. cerevisiae, commonly known as the fermented yeast of bread and beer, is widely used in food production (Yvert et al., 2003). As the most studied genetic model species, the whole genome sequences of A. oryzae and S. cerevisiae are widely available (Machida et al., 2005;Wei et al., 2007).
The biosynthesis and degradation of microbial fatty acids is important during fermentation, A. oryzae and yeast always consumed and transformed lipids of soy during its fermentation (Fukushima et al., 1991). Fatty acid oxidation is mediated by fatty acyl-CoA and acyl-CoA Binding Protein (ACBP) (Wei et al., 2007). ACBP is widely expressed in eukaryotic organisms and pathogenic bacteria (Vock et al., 2006) where is participates in the intracellular transport of acyl-CoAs (Knudsen et al., 2000). Previous studies have characterized the functions of ACBP in A. oryzae and S. cerevisiae. In A. oryzae. ACBP (AoAcbp1) encodes a polypeptide of 365 amino acids that mediates fatty acid metabolism through its interaction with acyl coenzyme A, contributing to the flavor of soy sauce through the metabolism of esters (Bouyakdan et al., 2015). Recently, Kwon et al. (2017) identified another ACBP (AoAcbp2) from A. oryzae, was likely essential for growth. In Hao et al. (2016) indicated that AoAcbp1 has a higher affinity for Palmitoyl-CoA and Stearoyl-CoA, revealed that AoAcbp1 is a long-chain ACBP in A. oryzae. 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). Amino acid sequence homologies between AoAcbp2 with AoAcbp1 and ScAcbp are 12.6% and 26.4%, respectively. AoAcbp2, is widely conserved in fungi and AoAcbp1, is only seen in filamentous fungi, but not in yeasts (Kwon et al., 2017).
For further study of ACBP, prokaryotic expression and purify is critical. It is convenient for us to study the function, structure and other properties of proteins. Proteins with high purity, simple culture processes, rapid growth and low media costs, are frequently used for protein expression studies. During protein purification, tagged fusion proteins are used for convenience. Maltose-Binding Protein (MBP) is a periplasmic protein of E. coli involved in chemotaxis and the uptake of maltose sugars (Reichenwallner et al., 2013). MBP was inserted downstream of the malE gene, resulting in the expression of an MBP fusion (Duplay et al., 1984). Amylose resin can be used as an affinity matrix to purity the MBP tagged protein (Sambrook and Russell, 2006). His-tags contain 6tandem histidine residues that do not influence the solubility or biological function of target proteins (Vijayalakshmi, 2008). N-terminal His-tagged proteins can be purified using one-step Immobilized Metal-chelate Affinity Chromatography (IMAC) (Li et al., 2017;Wang et al., 2017). In past studies, when MBP was used as fusion protein, whether it would affect the function of ACBP, it was uncertainty. Thus, we selected two common tagged fusion proteins for the purification.
In this study, MBP-ACBP and His-tagged ACBP were produced to high purity for the assessment of their binding affinities using Microscale Thermophoresis (MST) technology. Our findings were in agreement with results obtained using ITC and NMR (Pagano et al., 2008). The Monolith NTTM Protein Labeling Kit permits NT-647-NHS labeling of small amounts of protein (2-20 mM). Respective K d values were calculated using the mess action equation via Nano Temper software from duplicate reads of triplicate experiments (Wienken et al., 2010). MST was shown to efficiently determine such weak interactions between small fragments and proteins in our system. We reproducibly performed experimental binding measurements using minimal amounts of protein and fragments (Gudim et al., 2017).

Cloned ACBP and Construction of the Expression Vector
AoAcbp1 was cloned from A. oryzae 3.042 and ScACBP was cloned from S. cerevisiae S288c. pMAL-c4x and Pet-28a vector were used to express recombinant AoAcbp1 and ScAcbp fusion proteins,. Vector Pet-28a(+) has a His-tag in its N-terminus and pMAL-c4x has an MBP in its N-terminus. Pet-28a(+) was used for the expression of the His-tagged AoAcbp1 and ScAcbp fusions. pMAL-c4x was used to produce MBP fusions of AoAcbp1 and ScAcbp.
We found that ACBP from A. oryzae and S. cerevisiae encoded proteins of 365 and 87 amino acids in length, respectively. Both proteins contained an ACBP domain, as predicted by the Pfam program (http://pfam.xfam.org). This domain accounted for only a small portion of AoAcbp1. According to predictions from the Pfam program, we expressed a protein that contained domain fragments. This was initially cloned by PCR from A. oryzae cDNA using the following primers: AoAcbp1-domain for 5 ＇ GAATTCATGTCGGACTCTGTGAGGTATGCGTCGC AGACGCC 3 ＇ , Ao Acbp1-domain Rev 5 ＇ AAGCTTCTAGGGGCATTTATGATACCTTGG 3 ＇ . Underlined sequences were attached for Multisite Gateway cloning. The fragment was ligated to pMAL-c4x to select single clones, confirmed by diagnostic restriction analysis and PCR.

Recombinant Protein Expression
E. coli is used for recombinant protein expression as it does not inherently secrete proteins into the extracellular environment (Sommer et al., 2009). We constructed a series of expression vectors to facilitate extracellular protein production and purification.

Purification of Fusion-Tagged Proteins
For purification, cultured cells centrifuged at 12000 rpm for 20 min and resuspended in binding buffer on ice. Cells were sonicated for 10 min, centrifuged at 12000 rpm for 20 min and supernatants were collected. Recombinant proteins were purified using affinity chromatography columns.
Pet-AoAcbp1, Pet-ScAcbp have His-fusions with affinity for adsorption on Ni-NTA columns. His-tagged proteins remained bound to the column in the presence of washing buffer and were eluted by buffer containing 250 mM imidazole. Purified proteins were confirmed by SDS-PAGE and Coomassie blue staining.

Microscale Thermophoresis Binding Assays
The use of the Monolith NT TM Protein Labeling Kit, provides a convenient method of labeling purified AoAcbp1 and ScAcbp. Column A (for buffer exchange) permits protein purification in a buffer that lacks contain primary amines or imidazole. Proteins were mixed with NT-647-NHS dye 1:1 ratio (200 µL final volume) and incubated for 30 min at room temperature in the dark. Column B (gravity flow column for purification) was used to remove unbound dye and eluted fractions were assessed for fluorescent intensity suing the Monolith device. For this assays, 16 serial dilutions of the sample were produced and diluted 1:1 with fluorescently labeled protein. Sample were mixed for 5 min at room temperature and loaded into the NanoTemper glass capillaries to initiate MST analysis in a NanoTemper monolith NT.115 (California USA).
As a control, we expressed Pmal-c4x without ACBP and identically to pMAL-ScAcbp, to obtain MBP, verified whether MBP has affinity with each fatty-acids.

Results and Discussion
Cloning of the ACBP from A. oryzae and S. cerevisiae AoAcbp1 from A. oryzae 3.042 was 77% identical to A. fumigatus, A. clavatus, A. nomius and 99% identical to A. flavus (Yao et al., 2016). AoAcbp1 (GenBank accession number Ao3042_01296) was cloned and expressed as a MBP-AoAcbp1 fusion (purified by a step gradient of maltose) and a His-AoAcbp1 fusion (purified with a step gradient of imidazole). GFP-tagged AoAcbp1 localizs to punctate structures in the cytoplasm and is transported to vacuoles via the autophagy machinery (Kawaguchi et al., 2016). AoAcbp2 displays diffuse cytoplasmic staining (Kwon et al., 2017). ScAcbp cDNA (GenBank accession number YGR037C) from S. cerevisiae S288c, a highly conserved sequence (Gaigg et al., 2001), was cloned and expressed as an MBP-ScAcbp fusion and His-ScAcbp fusion and purified as described for AoAcbp1. Multiple alignment of the AoAcbp1 domain from several species revealed its importance to acyl-CoA binding activity, particularly Tyr and Lys residues which are evolutionarily conserved . Fragments containing the AoAcbp1-domain were confirmed by sequencing analysis (Sangon Corporation).

Prokaryotic Expression and Protein Purification
The expression vector was transformed into E. coli Rosetta (DE3) for protein expression. Bacteria were induced under varying temperatures and IPTG concentrations to ensure optimal purification conditions, confirmed by SDS-PAGE. Induction with 0.5 mM IPTG at 18°C produced the highest levels of recombinant proteins expression. To purify MBP tagged proteins, crude samples were loaded into amylose resin chromatography columns which were eluted with washing buffer. A Niagarose resin column was used for and His-tagged fusion proteins. Purified fusions were analyzed by SDS-PAGE.

Microscale Thermophoresis Binding Assays
Gravity flow and spin columns were used to purify the labeled proteins. MST analysis revealed that MBP-AoAcbp1 and His-AoAcbp1 had a binding preference for Stearoyl-CoA and Eicosanoyl-CoA (Table 1) (K d values and response curves are shown in Fig. S1 and S2) and the linear relativity shown that K d values (MBP-AoAcbp1 and His-AoAcbp1 with each fatty acyl-CoA) had no significant difference and had good linear correlation ( Fig. 2A). MBP-ScAcbp and His-ScAcbp had a binding preference for Myristoyl-CoA and Palmitoyl-CoA (Table 2) (K d values and response curves are shown in Fig. S3 and Fig. S4) and the linear relativity shown that K d values (MBP-ScAcbp and His-ScAcbp with each fatty acyl-CoA) had no significant difference and had good linear correlation (Fig. 2B). In addition, from the K d values (C4-C20), the presence of MBP or the His-tag did not influence the acyl-CoA binding affinity. The MBP-AoAcbp1-domain bound strongly to fatty acyl-CoA (C12:0-C18:0) (Fig. 3). As a control, MBP had no binding affinity with fatty acyl-CoA (C12:0-C18:0) (Fig. 4).     The MST data revealed that AoAcbp1 has a high specificity and affinity for long-chain acyl-CoA esters. Thus AoAcbp1 is a long chain ACBP. Furthermore, no binding was detected for each fatty acyl-CoA to the MBP tag , demonstrating that binding is AoAcbp1 specific. AoAcbp1 and ScAcbp had a preference for binding relatively long chain acyl-CoAs, that conform to the fatty acid composition of A. oryzae 3.042 and S. cerevisiae S288c.
In summary, we provide insight into the binding affinity and specificity of AoAcbp1 and ScAcbp for fatty acyl-CoA. Comparable affinity was observed between MBP and Histags of ACBP from A. oryzae and S. cerevisiae. In addition, no non-specific binding of the inserted tags was observed. To date, protein crystallization typically employs Histagged fusions for protein purification. As no influence of the tags was observed, MBP-tagged fusions are also applicable for crystallography studies and do not influence.