Micro-bioreactor cultivations of Fab producing Escherichia coli reveal genome-integrated systems as suitable for prospective studies on direct Fab expression effects.

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli and the high degree of similarity within this protein class, we are far from a generic platform technology. Indeed, feasible production of new Fab candidates remains challenging. In this study, we established a set-up that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated systems. Among the multitude of factors that influence Fab expression, we varied the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest. The resulting 32 production clones were characterized in carbon-limited micro-bioreactor cultivations with yields of 0 - 7.4 mg Fab/g cell dry mass. Antigen-binding region variations had the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performed better than the BL21(DE3) strain. Translocation mechanism variations mainly influenced leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the gene of interest integrated into the chromosome, reached Fab yields in the range of 80 to 300% of plasmid-based counterparts. Consequently, thegenome-integrated Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions. This article is protected by copyright. All rights reserved.

[1]  P. Neubauer,et al.  Amplification of ColE1 related plasmids in recombinant cultures of Escherichia coli after IPTG induction. , 1998, Journal of biotechnology.

[2]  Gerald Striedner,et al.  Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions , 2013, Applied and Environmental Microbiology.

[3]  L. Jermutus,et al.  Expression of high-affinity human antibody fragments in bacteria , 2012, Nature Protocols.

[4]  Feras Hatahet,et al.  Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli , 2010, Microbial cell factories.

[5]  M. Ellis,et al.  Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments , 2017, Biotechnology progress.

[6]  M. Ehrmann,et al.  Genetic Analysis of 15 Protein Folding Factors and Proteases of the Escherichia coli Cell Envelope , 2012, Journal of bacteriology.

[7]  Gerald Striedner,et al.  Preventing T7 RNA polymerase read-through transcription-A synthetic termination signal capable of improving bioprocess stability. , 2015, ACS synthetic biology.

[8]  B. Strukelj,et al.  Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells , 2013, Journal of Industrial Microbiology & Biotechnology.

[9]  K. Bayer,et al.  Fed-batch like cultivation in a micro-bioreactor: screening conditions relevant for Escherichia coli based production processes , 2015, SpringerPlus.

[10]  A. Lawson,et al.  A plasmid system for optimization of Fab' production in Escherichia coli: importance of balance of heavy chain and light chain synthesis. , 2002, Protein expression and purification.

[11]  Oliver Spadiut,et al.  Microbials for the production of monoclonal antibodies and antibody fragments , 2014, Trends in biotechnology.

[12]  Gerald Striedner,et al.  Plasmid‐free T7‐based Escherichia coli expression systems , 2010, Biotechnology and bioengineering.

[13]  D. Court,et al.  Recombineering: a homologous recombination-based method of genetic engineering , 2009, Nature Protocols.

[14]  B. Bukau,et al.  Prevention and reversion of protein aggregation by molecular chaperones in the E. coli cytosol: implications for their applicability in biotechnology. , 2002, Journal of biotechnology.

[15]  John C Joly,et al.  High‐level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple‐mutant (degP prc spr) host strain , 2004, Biotechnology and bioengineering.

[16]  Frank Kensy,et al.  The baffled microtiter plate: Increased oxygen transfer and improved online monitoring in small scale fermentations , 2009, Biotechnology and bioengineering.

[17]  F. Hartl,et al.  Molecular chaperones in cellular protein folding. , 1995, BioEssays : news and reviews in molecular, cellular and developmental biology.

[18]  T. Katsuda,et al.  Production of antibody fragments in Escherichia coli. , 2012, Methods in molecular biology.

[19]  C. Fernández,et al.  Tuned Escherichia coli as a host for the expression of disulfide‐rich proteins , 2011, Biotechnology journal.

[20]  U. Reichl,et al.  Effects of a recombinant gene expression on ColE1-like plasmid segregation in Escherichia coli , 2011, BMC biotechnology.

[21]  Gary Walsh,et al.  Biopharmaceutical benchmarks 2014 , 2014, Nature Biotechnology.

[22]  F. Wuest,et al.  Improved soluble expression of a single-chain antibody fragment in E. coli for targeting CA125 in epithelial ovarian cancer. , 2014, Protein expression and purification.

[23]  L. Pearl,et al.  Expression of the Fabs of human auto-antibodies in Escherichia coli: optimization and determination of their fine binding characteristics and cross-reactivity. , 2001, Journal of molecular biology.

[24]  Michaela Gebauer,et al.  Engineered protein scaffolds as next-generation antibody therapeutics. , 2009, Current opinion in chemical biology.

[25]  S. Lim,et al.  Enhanced expression of soluble antibody fragments by low-temperature and overdosing with a nitrogen source. , 2018, Enzyme and microbial technology.

[26]  Lynette M. Smith,et al.  A step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in E. coli. , 2008, Protein expression and purification.

[27]  C. Emrich,et al.  SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm , 2012, Microbial Cell Factories.

[28]  Frank Kensy,et al.  Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates , 2009, Microbial cell factories.

[29]  H Zola,et al.  Increased yield and activity of soluble single-chain antibody fragments by combining high-level expression and the Skp periplasmic chaperonin. , 2001, Protein expression and purification.

[30]  S. Dübel,et al.  SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli. , 2008, New biotechnology.

[31]  A. Marco.  Biotechnological applications of recombinant single-domain antibody fragments , 2011 .

[32]  R. Jalalirad.  Production of antibody fragment (Fab) throughout Escherichia coli fed-batch fermentation process: Changes in titre, location and form of product , 2013 .

[33]  Pratyoosh Shukla,et al.  Microbial platform technology for recombinant antibody fragment production: A review , 2017, Critical reviews in microbiology.

[34]  L. Schwimmer,et al.  Enhancement of antibody fragment secretion into the Escherichia coli periplasm by co-expression with the peptidyl prolyl isomerase, FkpA, in the cytoplasm. , 2013, Journal of immunological methods.

[35]  M. Camps.  Modulation of ColE1-like plasmid replication for recombinant gene expression. , 2010, Recent patents on DNA & gene sequences.

[36]  D. Humphreys,et al.  Production of Antibody Fab′ Fragments in E. coli , 2009 .

[37]  P. Neubauer,et al.  Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures , 2013, Microbial Cell Factories.

[38]  Johannes Buchner,et al.  How antibodies fold. , 2010, Trends in biochemical sciences.

[39]  J. Choi,et al.  Secretory and extracellular production of recombinant proteins using Escherichia coli , 2004, Applied Microbiology and Biotechnology.