title: Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory.
abstract: BACKGROUND
  Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs.
  
  
  RESULTS
  We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host.
  
  
  CONCLUSIONS
  Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve additional increases in the yields of scFvs.
authors.affiliation: Environmental Microbiology Laboratory, Helmholtz Centre for Infection Research, Inhoffenstr, 7, 38124 Braunschweig, Germany. thorben.dammeyer@helmholtz-hzi.de
authors.forename: Thorben
authors.initials: T
authors.lastname: Dammeyer
authors.forename: Miriam
authors.initials: M
authors.lastname: Steinwand
authors.forename: Sarah-C
authors.initials: SC
authors.lastname: Krüger
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.forename: Michael
authors.initials: M
authors.lastname: Hust
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Timmis
mesh_terms: D000595:Amino Acid Sequence; D002097:C-Reactive Protein / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000276:immunology; D019897:Periplasm / Q000378:metabolism; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012995:Solubility
chemical_list: D018396:Mucin-1; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D002097:C-Reactive Protein