title: Production of Recombinant Peanut Allergen Ara h 2 using Lactococcus lactis.
abstract: BACKGROUND
  Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis.
  
  
  RESULTS
  A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2.
  
  
  CONCLUSIONS
  Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.
authors: Bioneer A/S, DK-2970 Hørsholm, Denmark. jag@bioneer.dk.
authors: Jacob
authors: J
authors: Glenting
authors: Lars K
authors: LK
authors: Poulsen
authors: Kentaro
authors: K
authors: Kato
authors: Søren M
authors: SM
authors: Madsen
authors: Hanne
authors: H
authors: Frøkiær
authors: Camilla
authors: C
authors: Wendt
authors: Helle W
authors: HW
authors: Sørensen
references: Methods Mol Biol. 1995;47:195-9
references: 7550735
references: Appl Microbiol Biotechnol. 1992 May;37(2):216-24
references: 1368776
references: Protein Expr Purif. 1997 Feb;9(1):33-9
references: 9116499
references: Arch Biochem Biophys. 1997 Jun 15;342(2):244-53
references: 9186485
references: J Allergy Clin Immunol. 1999 Apr;103(4):559-62
references: 10200001
references: Nat Med. 1999 Apr;5(4):387-91
references: 10202926
references: J Allergy Clin Immunol. 1999 Nov;104(5):969-77
references: 10550741
references: Science. 2000 Aug 25;289(5483):1352-5
references: 10958782
references: Appl Environ Microbiol. 2002 Oct;68(10):5051-6
references: 12324356
references: Infect Immun. 2003 Apr;71(4):1887-96
references: 12654805
references: Cochrane Database Syst Rev. 2003;(2):CD002893
references: 12804442
references: Microbiology. 2003 Aug;149(Pt 8):2193-201
references: 12904559
references: Protein Expr Purif. 2003 Oct;31(2):250-9
references: 14550644
references: Vaccine. 2003 Dec 8;22(1):87-95
references: 14604575
references: Allergy. 2004 Jan;59(1):45-53
references: 14674933
references: Methods. 2004 Mar;32(3):313-20
references: 14962766
references: Clin Exp Allergy. 2004 Apr;34(4):583-90
references: 15080811
references: Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M. 2003;(94):97-105; discussion 106
references: 15119026
references: J Allergy Clin Immunol. 2004 Jun;113(6):1013-23; quiz 1024
references: 15208577
references: Immunol Lett. 2005 Sep 15;100(2):153-8
references: 15899521
references: Mol Nutr Food Res. 2005 Oct;49(10):963-71
references: 16189800
references: Biochem J. 2006 May 1;395(3):463-72
references: 16372900
references: Clin Gastroenterol Hepatol. 2006 Jun;4(6):754-9
references: 16716759
references: Expert Rev Mol Med. 2007;9(1):1-18
references: 17210088
references: Microb Cell Fact. 2007;6:12
references: 17417967
references: Clin Exp Allergy. 1998 Aug;28(8):984-91
references: 9756203
references: Mol Microbiol. 1999 Apr;32(1):75-87
references: 10216861
references: Allergy. 1999 Apr;54(4):380-5
references: 10371098
references: J Allergy Clin Immunol. 2000 Sep;106(3):409-18
references: 10984358
references: Ann Allergy Asthma Immunol. 2002 Feb;88(2):183-9
references: 11868923
references: Microb Cell Fact. 2005 Sep 6;4:26
references: 16144545
references: Appl Environ Microbiol. 1993 Aug;59(8):2730-3
references: 16349028
references: Immunol Allergy Clin North Am. 2006 May;26(2):191-206, v-vi
references: 16701140
references: J Allergy Clin Immunol. 2006 Aug;118(2):434-40
references: 16890769
references: FEBS J. 2006 Oct;273(19):4425-34
references: 16956374
references: Glycobiology. 2007 Feb;17(2):220-30
references: 17095532
references: J Allergy Clin Immunol. 2007 Apr;119(4):826-30
references: 17335886
references: Mol Immunol. 1996 Mar-Apr;33(4-5):399-405
references: 8676891