ARTICLE pubs.acs.org/Biomac
Endotoxin Removing Method Based on Lipopolysaccharide Binding Protein and Polyhydroxyalkanoate Binding Protein PhaP Jian Li,†,‡ Guanguan Shang,†,‡ Mingliang You,‡ Siwu Peng,‡ Zhihui Wang,‡ Hongning Wu,‡ and Guo-Qiang Chen*,§ ‡
Multidisciplinary Research Center, Shantou University, Shantou 515063, Guangdong, China Department of Biological Science and Biotechnology, School of Life Science, Tsinghua University, Beijing 100084, China
§
ABSTRACT: Polyhydroxyalkanoates (PHAs) granule associated protein PhaP has a strong affinity to PHA and other hydrophobic polymers. Human lipopolysaccharide binding protein (hLBP) is a natural endotoxin receptor in plasma. In this study, genes encoding hLBP fused with PhaP were expressed in Pichia pastoris GS115 for production of the fusion protein. The purified rhLBP-PhaP fusion protein was immobilized on particles of polyhydroxybutyrate (PHB), which is a member of microbial polyhydroxyalkanoates (PHA). The rhLBPPhaP-coated PHB particles were added to endotoxin containing water and protein solutions to study their endotoxin removal and protein recovery efficiencies. The influences of ionic strengths and pH on endotoxin removal and protein recovery in different protein solutions were also studied using acidic proteins including bovine serum albumin (BSA), ovalbumin, and basic protein R-chymotrypsinogen as model proteins. The results showed that rhLBP-PhaP particles could remove endotoxin with an efficiency of over 90%. All endotoxin removal and protein recovery efficiencies were only slightly affected by ionic strengths but were drastically affected by pH changes. Our results demonstrated that rhLBP-PhaP particles with their high efficiency, ease of preparation, and nontoxicity will be a suitable system for endotoxin removal in the protein purification industry.
’ INTRODUCTION Endotoxin, also called pyrogen, is an integral outer membrane component derived from gram-negative bacteria such as Escherichia coli or Pseudomonas spp.1,2 Endotoxin is mainly lipopolysaccharides (LPS). LPS can tolerate high temperature and pH changes and they show strong biological effects, even at a very low concentration, in human and many other animals.3,4 For example, LPS induced pyrogenic and endotoxic shocks after intravenous administration even with less than 1 ng/kg in human.5 According to pharmacopoeias, the general threshold level of endotoxin is set at five endotoxin units (EU) per kg body weight per hour for intravenous administration, while 1 EU is equal to only 100 pg bacterial endotoxin.3,6 Hence, the development of techniques to remove endotoxins from products used for intravenous administration is crucial, especially for those therapeutic protein solutions produced from recombinant microorganisms. Various methods have been developed to achieve this goal for different target solutions.7 Some conventional methods have been used for endotoxin removal from nonprotein solutions and have achieved good results, such as ion-exchange chromatography, ultrafiltration, two-phase extraction, and sucrose gradient r 2011 American Chemical Society
centrifugation.7 However, the removal of endotoxin from a protein containing solution in a biorelated system is extremely difficult using the classical methods; the difficulty comes from the low protein recovery after the LPS removal process.8 Recently, the affinity adsorption method has been developed for endotoxin removal, and it has been proven to be unique and highly effective.1 These techniques allow a purification process based on biological functions rather than individual physical or chemical properties.1,9 Traditionally, for endotoxin removal, affinity chromatography uses a soft gel as the matrix, virulent cyanogen bromide as an activation agent, and histamine or polymyxin B as the ligand.10,11 Although these have been employed, some shortcomings still exist for large scale applications, such as the fragile soft gels, the high cost and complexity to immobilize ligands on matrix, and the toxicity of ligands.9 Recently, affinity membranes have received attention due to their low circulation resistance although the introduction of a ligand and spacing arm is complicated, and a specific membrane module is needed for practical Received: October 14, 2010 Revised: November 24, 2010 Published: January 24, 2011 602
dx.doi.org/10.1021/bm101230n | Biomacromolecules 2011, 12, 602–608
Biomacromolecules
ARTICLE
application.12,13 The above results suggest that improved affinity adsorbents for endotoxin removal can be achieved from improved ligands as well as their supports. Human lipopolysaccharide-binding protein (hLBP) is a natural endotoxin receptor existing in normal serum at
