An efficient depyrogenation method for recombinant bacterial outer membrane lipoproteins

https://doi.org/10.1016/j.pep.2014.02.012Get rights and content

Highlights

  • OprI-antigen fusions were cloned and purified from the E. coli outer membranes.

  • Detergents and solid phase adsorbents were ineffective to solve LPS contamination.

  • A phenol-based extraction was used to clear LPS from the outer membrane preparations.

  • Recombinant lipoproteins were thus consistently purified with less than 0.02 EU/μg.

  • The purified recombinant lipoproteins could stimulate dendritic cells.

Abstract

Bacterial outer membrane lipoproteins are anchored in the outer membrane lipid layer in close association with lipopolysaccharides (LPS) and with other hydrophobic membrane proteins, making their purification technically challenging. We have previously shown that a thorough delipidation of outer membrane preparations from the Escherichia coli expression host is an important step to eliminate contaminant proteins when purifying recombinant antigens expressed in fusion with the Pseudomonas aeruginosa OprI lipoprotein. Here we report the cloning and expression of three antigens in fusion with OprI (ovalbumin, eGFP and BbPDI) and our efforts to deal with the variable LPS contamination levels observed in different batches of purified lipoproteins. The use of polymyxin B columns or endotoxin removal polycationic magnetic beads for depyrogenation of purified lipoproteins resulted in high protein losses and the use of Triton X-114 or sodium deoxycholate during the course of affinity chromatography showed to be ineffective to reduce LPS contamination. Instead, performing a hot phenol/water LPS extraction from outer membrane preparations prior to metal affinity chromatography allowed the purification of the recombinant fusion lipoproteins with LPS contents below 0.02 EU/μg of protein. The purified recombinant lipoproteins retain their capacity to stimulate bone marrow-derived dendritic cells allowing for the study of their immunomodulatory properties through TLR2/1. This is a simple and easy to scale up method that can also be considered for the purification of other outer membrane lipoproteins.

Introduction

Molecules from microbial origin have long been used experimentally as modulators of immune responses with therapeutic and prophylactic purposes [1], [2]. Recently, the understanding of the molecular basis underlying their immunomodulatory properties experienced important advances with the discovery of the pattern recognition receptors (PRRs)1 [3]. PRRs, such as Toll-like receptors (TLRs), are a series of innate receptors that recognize conserved components of microorganisms, in most cases cell wall elements or nucleic acids with microbe specific features, globally called pathogen associated molecular patterns (PAMPs) [3]. At present, most of the novel strategies for the development of subunit vaccines rely on the study of how PAMPs stimulate innate immunity through PRRs and on the understanding of how this activation shapes the subsequent adaptive immune responses [4], [5], [6].

Based on previously described cloning plasmids engineered for the production of immunogenic formulations [7] and for the identification and characterization of antigens [8], we have recently developed a new system for cloning and expression of antigens in fusion with the OprI lipoprotein from the outer membrane of Pseudomonas aeruginosa [9]. The system enables the insertion of OprI-antigen fusions, with the typical structure of a TLR2/1 ligand, in the outer membrane of the Escherichia coli expression host from where it is possible to obtain immunogenic formulations with different capacity to stimulate innate immunity. These are: (1) outer membrane vesicles, incorporating the recombinant lipoproteins and released during the period of protein expression; (2) outer membrane fragments, containing the recombinant lipoproteins together with other membrane proteins and lipopolysaccharides (LPS) obtained from the outer membrane by solubilisation in native conditions with octyl β-d-glucopyranoside (OG) detergent followed by affinity chromatography; (3) OprI-antigen fusion proteins, purified by denaturing affinity chromatography after trichloroacetic acid (TCA)/acetone delipidation of outer membrane [9]. This purification procedure proved to be sufficient to obtain the OprI-antigen fusions free from contaminant proteins. However, in the laboratory routine, we have detected variable levels of the endotoxin LPS when producing repeated batches of the same recombinant lipoproteins. An important application of the system is to profit from the single effect of a physical link between antigens and the TLR2/1 ligand OprI. So, variable or high LPS content in those immunogens is detrimental since it is a TLR4 ligand and thus alters the predominant TLR2/1 nature of the stimulus. Bacterial outer membrane lipoproteins are expressed in the cytoplasm in the form of a prolipoprotein with an N-terminal signal peptide and are then translocated across the inner membrane to be processed to the mature form in the periplasmic side. During this process the signal peptide is cleaved, the triacylated moiety is acquired and the mature lipoproteins are transported and anchored in the outer membrane [10], [11]. Since the triacylated mature form, recognised by TLR2/1, is only present in the outer membrane of the bacterial cell [12], where it is in close association with LPS, the purification protocol has to specifically address the LPS removal from the preparations.

Considerable efforts have been made to settle methods for efficient endotoxin removal from protein solutions, including chromatography with solid phase adsorbents like polymyxin B, ion-exchange chromatography, aqueous two-phase micellar extraction with Triton X-114 or size exclusion of endotoxin molecules by gel filtration chromatography, ultra-filtration or sucrose gradient centrifugation [13], [14], [15], [16]. In this work we tested polymyxin B columns, endotoxin removal polycationic magnetic beads and modifications in the affinity chromatography protocols to reduce the LPS contamination in three OprI fusion lipoproteins. These methods proved to be unsuccessful and we propose a hot-phenol/water LPS extraction from outer membrane preparations prior to affinity chromatography as a new strategy. With this protocol we have consistently obtained recombinant OprI fusion antigens with LPS contents below 0.02 endotoxin units (EU)/μg of protein without affecting their biological activity.

Section snippets

Cloning of Ova, eGFP and BbPDI coding sequences in the OprI based vector pOLT7

Coding sequences from the chicken ovalbumin (Ova), enhanced green fluorescent protein (eGFP) and Besnoitia besnoiti protein disulfide isomerase (BbPDI) were cloned in our plasmid pOLT7 [9], in the EcoRI and XhoI sites of its multiple cloning site to produce recombinant proteins in fusion with an N-terminal OprI lipoprotein.

The Ova sequence was obtained from the magnum region of the oviduct of a chicken (Gallus gallus domesticus) in the egg laying phase. Total RNA was isolated (High Pure RNA

Cloning, expression and purification of OprI fusion antigens

We have cloned coding sequences from two model antigens widely used in experimental immunology, the ovalbumin (Ova) and the eGFP, and an antigen from the apicomplexan parasite B. besnoiti, the protein disulfide isomerase (BbPDI) [22]. The three sequences were cloned in our previously described plasmid pOLT7, which enables the expression of these antigens in fusion with an N-terminal lipidated OprI lipoprotein. The correct sequences of the recombinant plasmids, pOLT7-OVApx, pOLT7-eGFP and

Conclusions

The close association between outer membrane lipoproteins and LPS in the Gram-negative bacteria and the chemical similarities shared by their lipid moieties make their separation difficult during purification procedures. To remove LPS from recombinant antigens expressed as fusions with the OprI lipoprotein the polymyxin B columns, endotoxin removal polycationic magnetic beads and the use of Triton X-114 or sodium deoxycholate during the course of affinity chromatography have shown to be

Acknowledgments

We are thankful to I.M. Cheeseman for the use of pIC113 plasmid. This work was supported by Fundação para a Ciência e a Tecnologia, Portugal: project Grants PTDC/CVT/113889/2009 and PTDC/CVT/65674/2006; research contract by the Ciência 2007 Program (D.M. Santos); PhD fellowship SFRH/BD/31445/2006 (E. Marcelino).

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