CLONING AND RECOMBINANT EXPRESSION OF NOVEL HYDROPHOBIC LIGAND BINDING PROTEIN (HLBP) FROM CESTODES

Abstract

Parasitic helmiths have evolved adaptations in cellular metabolism in order to survive under low oxygen tension in their respective hosts. One unique physiological feature is the down regulation of enzymes responsible for the synthesis of hydrophobic metabolites (usually requiring oxygen at some stage in their synthetic pathway). For example, adult parasitic helminths do not synthesise long-chain fatty acids, cholesterol or related sterols and rely on uptake from the host. Therefore, it is essential for these helmiths to express an efficient hydrophobic binding system to aid the uptake, storage and intracellular transport of these important metabolic substrates. A new family of abundant intracellular hydrophobic ligand binding proteins (HLBPs) from cestodes has been characterised with respect to its biochemistry. We report now on the cloning of cestode HLBPs and we describe their sequence relationship to lipid binding proteins and acyl CoA binding proteins. Progress towards the expression and purification of recombinant cestode HLBP for structural and mutagenesis studies will also be discussed.

Introduction

Hydrophobic ligand binding proteins (HLBP's) are a widely distributed group of low molecular weight (14-16kDa) proteins that can be found in the plasma membrane and extracellular fluids. Members of the HLBP family include adipocyte and ileal- binding proteins, myelin P2 protein and several fatty acid binding proteins (FABP's), cellular retinol-binding proteins and retinoic acid-binding proteins. Although these proteins have been studied extensively, their biological role is still not fully understood. However some of their main functions are thought to include:- fatty acid uptake, transfer and storage; targeting of fatty acids to specific organelles and pathways; sequestration of toxic compounds; and intracellular transport and metabolism of small hydrophobic molecules. Novel HLBP's, found in high concentration (5%) in the cytosol, have been isolated from Moniezia expansa (Janssen and Barrett, 1995) and Hymenolepis diminuta (Saghir, 1998). From mass spectra data they have been found to be of a lower molecular weight (8kDa) than other HLBP's with limited sequence similarity to other proteins. In their native form they are found to bind saturated and unsaturated fatty acids but not their Co-A derivatives, sterols and several anthelmintic agents, such as bithionol, hexachlorophene and niclosamide (Janssen and Barrett, 1995; Saghir, 1998).

Attempts to express previously sequenced HLBP genes from Moniezia expansa and Hymenolepis diminuta in Pet23d expression vectors (Novagene) met with failure due to degradation of the recombinant protein (Fig1). Dideoxysequencing of the original cloning vectors pUC18 (Gibco), produced the DNA and translated protein analysis (Fig 2;3). Insertion of the M. expansa HLBP gene into a pGex-4T-3 vector (Pharmacia Biotech) and subsequent transformation into E.coli BL21-, produced a soluble fusion protein with a thrombin cleavage site when induced with 0.5mM IPTG (Fig 4). Purification of the fusion protein was by affinity chromatography (Fig 5). Optimisation of thrombin cleavage was found to require an increase in the concentration of the protein cleaving enzyme by as much as twice that recommended by the supplier (Pharmacia Biotech) with a reaction time of 24 hours at 22 degrees Centigrade. Separation of GST and HLBP was by affinity chromatography (Fig 6). Both proteins were concentrated by Centricon spin columns (Millipore) with a cut off of 10kD. Concentrated proteins were run on 15% SDS Page and confirmed by Western Blot (Fig 8). Protein assays estimate 9 mg of fusion protein was produced from 1 litre of culture grown under optimal conditions (Fig 4), from this, 2mg of HLBP was produced.

Cloning and Expression

HLBP from both Moniezia expansa and Hymenolepis diminuta have previously been cloned by anchor based PCR. Ligation into pUC18 cloning vectors (Barrett et al, 1997) and subsequent sequencing was followed by the insertion of both genes into high expression Pet 23d vectors, under an IPTG inducible T7 promoter in E. coli BL21(DE3) unpublished however all attempts at expression were unsatisfactory with degradation of the recombinant proteins. Primers were designed and PCR was used to create the Moneizia expansa HLBP gene with EcoR1 and Xho1 restriction sites (Fig 1). These were then used for the correct insertion of the gene into a pGEX-4T-3 (Pharmacia Biotech) Glutathione-S-transferase (GST) gene fusion vector. This vector under the tight regulation of the IPTG inducible tac promoter, allows for the production of soluble GST-HLBP fusion protein, linked by a thrombin cleavage site (Chang, 1985). The gene itself is shown here in blue, whereas the specially designed forward and reverse PCR primers are indicated in black. The red sequences are complement which are lost after digestion with the appropriate restriction enzymes.

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Figure 1. Moneizia expansa HLBP gene sequence together with restriction sites for insertion into pGex-4T-3 expression vector.

Translation

The translation of the Monezia expansa HLBP gene sequence (Fig 2). The colour scheme for amino acids follows the "Blue is for Basic" colour scheme suggested in the BBN Systems and Technologies analysis software Prophet.

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Figure 2. The translation of the Monezia expansa HLBP gene sequence.

Sequence Alignment

Fig 3 shows the protein sequence alignment of translated HLBP genes cloned from Hymenolepis diminuta (A) and Moniezia expansa (B) showing highly conserved areas. Conserved residues are shown with a dot. A black dot signifies an identical amino acid at an identical location along the peptide chain. A red dot signifies a similar type of amino acid at an identical location along the peptide chain. Colour codes and abbreviations are as in Fig 2. Preliminary analysis of secondary structure shows the HLBP to consist predominantly of alpha helical structures, unlike Fatty acid binding proteins, the structure of which is largely of a beta sheet configuration (Fig 9).

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Figure 3. The protein sequence alignment of translated HLBP genes cloned from Hymenolepis diminuta (A) and Moniezia expansa (B). See text for details.

Expression of Recombinant Protein

Optimisation of expression was initially thought unnecessary, and the host cells were incubated at 37^oC in LB containing 100mg/ml ampicilin. In an attempt to avoid the formation of inclusion bodies, lower temperatures of 32^oC and 30^oC were employed. Results show that higher concentrations of fusion protein per unit of optical density are produced at the lower temperatures. This is confirmed by work carried out by Pharmacia Biotech (Saluta and Bell, 1999). Studies (Fig 4) on the effect of induction with IPTG on the growth curve of a culture of BL21/pGex-4T-3/MexHLBP, show detrimental effects when a culture is induced too early (less than 2.0 O/D at 600nm). Likewise a culture that is allowed to continue to grow after IPTG has been degraded will show a deleterious effect as the bacterium recovers and degrades the recombinant protein. For optimum expression, the cultures were allowed to grow to an O/D at 600nm =2.0 and then induced with 0.5mM IPTG for 4hrs before being harvested.

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Figure 4. Expression of the recombinant protein.

Purification of GST Fusion Protein

A one litre culture was grown under optimal conditions and harvested for the soluble fraction (see Fig 4). This was then passed through an affinity column containing glutathione agrose (Sigma chemicals), which had previously been equilibrated with 20mM potassium phosphate buffer pH 7.0. The bound affinity column was then washed with the phosphate buffer, before being eluted with 50mM Tris-HCL pH 8.8 containing 25mM reduced glutathione (Sigma) and collected in 1ml fractions. The fractions were then assayed for both protein content and GST activity (Fig5). Fractions that contained GST activity were then pooled for cleavage with thrombin (2 units /100mg fusion protein at 22^oC for 24 hrs).

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Figure 5. Purification of GST fusion protein.

Separation of GST and HLBP

Following digestion with thrombin the fusion proteins now cleaved, were passed through a second affinity column containing equilibrated glutathione agrose. Fractions were collected immediately in 1ml volumes. The column was washed as before with 20mM phosphate buffer pH7.0. The flow through being collected in 1ml fractions. The GST was eluted with Tris-HCL pH 8.8 containing 25 mM reduced glutathione, and collected in 1ml fractions. All fractions were assayed for protein activity (Fig 6). Samples containing like proteins were pooled and run on SDS page. and Western Blot Fig 8.

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Figure 6. Separation of GST and HLBP.

Activity of HLBP

Preliminary indications of the activity of the recombinant HLBP are encouraging. From fluorescence studies to date HLBP is clearly seen to bind ANS with an increase in fluorescence (Ex max 360nm-Em max 490nm) in a protein concentration dependent manor (Fig 7). Furthermore similar tests with Schistosoma japonicum GST show no binding of ANS Fig 7, ruling out increased fluorescence from any residual GST, which, although being a very small percentage of the total protein content, could never the less, have influenced the results.

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Figure 7. Activity of HLBP.

SDS Page and Western Blot

Fig 8 shows silver staining of the 15% SDS Page clearly shows the recombinant M. expansa HLBP. The protein can often diffuse and is sometimes lost during treatment with acetic acid and methanol following staining with coommassie blue. Both the fusion protein and the efficiency of thrombin to cleave the fusion protein are also clearly visible on this gel. The Western blot onto nitrocellulose, reveals the high specificity of a M. expansa HLBP antibody, which not only recognises the native and recombinant protein with high fidelity, but also the fusion protein whether purified or as part of a crude extract.

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Figure 8. SDS Page and Western Blot.

3D Images

Fig 9 contrasts the structures of the Intestinal Fatty Acid Binding Protein (FABP), seen here on the right, with that of the closest homologue to HLBP found to date. The Acyl Coenzyme A Binding Protein (pictured left) also consists mainly of alpha helices and is of a similar size to HLBP. Both protein structures are shown in the ribbon format and are colour coded according to structure. The pictures are from PDB files constructed from nmr data, and interpreted using Rasmol software.

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Figure 9. 3D images. Left: Acyl Coenzyme A Binding Protein; Right: Intestinal Fatty Acid Binding Protein.

Acknowledgements

This work was supported by the Wellcome Trust.

References

Barrett, J., Saghir, N., Timanova, A., Clarke, K., and Brophy, P.M., (1997). Characterisation and properties of an intracellular lipid-binding protein from the tapeworm Moniezia expansa. European Journal of Biochemistry, 250, 269-275.

Chang, J-Y. (1985). Thrombin specificity, requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. European Journal of Biochemistry, 151, 217-224.

Janssen, D., and Barrett, J., (1995). A novel lipid-binding protein from the cestode Moniezia expansa. Biochemical Journal, 311, 49-57.

Saghir, N., (1998). PhD thesis. UCW Aberystwyth.

Saluta, M., and Bell, P.A., (1998). Troubleshooting GST fusion protein expression in E.coli. Life Science News, 1, p15.