Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory

Abstract

BackgroundRecombinant 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.ResultsWe 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.ConclusionsPseudomonas 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.

Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression

“…The data above emphasize the value of environmental bacteria such as P. putida KT2440 as the starting point of a platform strain with a degree of robustness and metabolic capacities that cannot be delivered by other bacterial hosts [21,[26][27][28][29]. The judicious removal of just a limited number genes encoding undesirable traits has sufficed to upgrade the already useful properties of P. putida towards an optimal and standardized chassis for synthetic biology and metabolic engineering, especially in the cases where the pathways under study generate toxic intermediates and/or demand a biochemical background that supplies enough ATP and NADPH to sustain heterologous bioreactions.…”

“…On these bases, it does not come as a surprise that P. putida KT2440, a non-pathogenic strain of the soil bacterium P. putida is being increasingly used as a host for heterologous DNA expression for different biotechnological purposes [26][27][28]. This strain is not only certified as GRAS (generally regarded as safe [29]) and endowed with a remarkable metabolic versatility, but it also possesses a noteworthy tolerance to many organic compounds [28,30] and other stressful conditions, such as those that generate reactive oxygen species (ROS).…”

Synthetic gene design—The rationale for codon optimization and implications for molecular pharming in plants

“…Of these software packages, citations have been found for JCat and OPTIMIZER for expression systems such as E. coli (Fahimi et al 2016;Guo et al 2016;Karkhah & Amani 2016;Zhao et al 2016), S. cerevisiae (Ask et al 2013;Guadalupe-Medina et al 2013;Li et al 2015;Milne et al 2015;Solis-Escalante et al 2013), N. benthamiana (Binder et al 2014), HEK293 cells (Shah et al 2015), B. subtilis (Reilman et al 2014), C. crescentus (Ko et al 2013), P. putida (Dammeyer et al 2013;Dammeyer et al 2011), S.…”

Properties of alternative microbial hosts used in synthetic biology: towards the design of a modular chassis

“…coli strains. In that sense, the synthesis of single-chain Fv fragments [55] as well as camel antibodies (nanobodies) [56] has been achieved in P. putida with good yields and proper folding. Apart from proteins, P. putida has been used to produce a wide number of small molecules such as rhamnolipids, terpenoids, polyketides and non-ribosomal peptides (recently reviewed in [57]).…”