Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort

A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.

[1]  Stefan Schillberg,et al.  A versatile coupled cell‐free transcription–translation system based on tobacco BY‐2 cell lysates , 2015, Biotechnology and bioengineering.

[2]  Shigeyuki Yokoyama,et al.  Structural Basis for Substrate Selection by T7 RNA Polymerase , 2004, Cell.

[3]  Ashty S. Karim,et al.  Enhancing control of cell-free metabolism through pH modulation , 2019 .

[4]  Scott A. Walper,et al.  Quantification of Interlaboratory Cell-free Protein Synthesis Variability. , 2019, ACS synthetic biology.

[5]  G. Church,et al.  Establishing a Cell-Free Vibrio natriegens Expression System. , 2018, ACS synthetic biology.

[6]  Christopher J. Nachtsheim,et al.  A Class of Three-Level Designs for Definitive Screening in the Presence of Second-Order Effects , 2011 .

[7]  Velia Siciliano,et al.  Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology. , 2020, ACS synthetic biology.

[8]  R. Gesteland,et al.  Unfolding of Escherichia coli ribosomes by removal of magnesium. , 1966, Journal of molecular biology.

[9]  Dong-Myung Kim,et al.  Prolonging cell-free protein synthesis with a novel ATP regeneration system. , 1999, Biotechnology and bioengineering.

[10]  M. Esmann,et al.  The Effect of Ionic Strength and Specific Anions on Substrate Binding and Hydrolytic Activities of Na,K-ATPase , 1997, The Journal of general physiology.

[11]  R. Zitomer,et al.  Magnesium dependence and equilibrium of the Escherichia coli ribosomal subunit association. , 1972, Journal of molecular biology.

[12]  F. Kawamura,et al.  Defect in the Formation of 70S Ribosomes Caused by Lack of Ribosomal Protein L34 Can Be Suppressed by Magnesium , 2014, Journal of bacteriology.

[13]  Tae-Wan Kim,et al.  Rapid production of milligram quantities of proteins in a batch cell-free protein synthesis system. , 2006, Journal of biotechnology.

[14]  F F Bier,et al.  Lab on a Chip Miniaturisation for chemistry , physics , biology , materials science and bioengineering , 2015 .

[15]  T. Lohman,et al.  Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity , 1978, Quarterly Reviews of Biophysics.

[16]  Ashty S. Karim,et al.  Controlling cell-free metabolism through physiochemical perturbations. , 2018, Metabolic engineering.

[17]  S. Kubick,et al.  A Continuous-Exchange Cell-Free Protein Synthesis System Based on Extracts from Cultured Insect Cells , 2014, PloS one.

[18]  T. Steitz,et al.  The contribution of metal ions to the structural stability of the large ribosomal subunit. , 2004, RNA.

[19]  Y Endo,et al.  A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Kaoru Kobayashi.  Summary of recombinant human serum albumin development. , 2006, Biologicals : journal of the International Association of Biological Standardization.

[21]  M. Jewett,et al.  Cell-free synthetic biology: thinking outside the cell. , 2012, Metabolic engineering.

[22]  Yutaka Hori,et al.  Cell-free extract based optimization of biomolecular circuits with droplet microfluidics. , 2017, Lab on a chip.

[23]  A. Spirin,et al.  Studies on the structure of ribosomes. II. Stepwise dissociation of protein from ribosomes by caesium chloride and the re-assembly of ribosome-like particles. , 1966, Journal of molecular biology.

[24]  Andrew Buchanan,et al.  Coping with complexity: Machine learning optimization of cell‐free protein synthesis , 2011, Biotechnology and Bioengineering.

[25]  G. Zubay,et al.  In vitro synthesis of protein in microbial systems. , 1973, Annual review of genetics.

[26]  Paul S. Freemont,et al.  Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria , 2018, Proceedings of the National Academy of Sciences.

[27]  T. Hunt,et al.  Preparation and use of nuclease-treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. , 1983, Methods in enzymology.

[28]  Jung-Won Keum,et al.  Prolonged cell‐free protein synthesis using dual energy sources: Combined use of creatine phosphate and glucose for the efficient supply of ATP and retarded accumulation of phosphate , 2007, Biotechnology and bioengineering.

[29]  Paul S Freemont,et al.  Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool. , 2017, Biotechnology journal.

[30]  R. Thiebe.  Aminoacylation of tRNA. Magnesium requirement and spermidine effect , 1975, FEBS letters.

[31]  Marshall W. Nirenberg,et al.  The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides , 1961, Proceedings of the National Academy of Sciences.

[32]  Stefan Schillberg,et al.  Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system , 2014, BMC Biotechnology.

[33]  Guillaume Lambert,et al.  Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components , 2016, Cell.

[34]  A. Spirin,et al.  Studies on the structure of ribosomes. 3. Stepwise unfolding of the 50 s particles without loss of ribosomal protein. , 1966, Journal of molecular biology.

[35]  A. Warshel,et al.  Calculations of electrostatic interactions in biological systems and in solutions , 1984, Quarterly Reviews of Biophysics.

[36]  B. Honig,et al.  Classical electrostatics in biology and chemistry. , 1995, Science.

[37]  Karen M Polizzi,et al.  Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform , 2019, Biotechnology and bioengineering.

[38]  Rui Gan,et al.  A combined cell-free transcription-translation system from Saccharomyces cerevisiae for rapid and robust protein synthe. , 2014, Biotechnology journal.

[39]  Ashty S Karim,et al.  High-Throughput Optimization Cycle of a Cell-Free Ribosome Assembly and Protein Synthesis System. , 2018, ACS synthetic biology.

[40]  Philip Ramsey,et al.  Efficient biological process characterization by definitive-screening designs: the formaldehyde treatment of a therapeutic protein as a case study , 2012, Biotechnology Letters.

[41]  Geoffrey Chang,et al.  The past, present and future of cell-free protein synthesis. , 2005, Trends in biotechnology.

[42]  Shelton S. Bradrick,et al.  A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes , 2013, RNA.

[43]  By Jared L Dopp,et al.  Cell-free supplement mixtures: Elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract. , 2019, Biotechnology advances.

[44]  Takashi Suzuki,et al.  Cell‐Free Protein Synthesis System Prepared from Insect Cells by Freeze‐Thawing , 2006, Biotechnology progress.

[45]  J. Swartz,et al.  Energizing cell-free protein synthesis with glucose metabolism. , 2005, Biotechnology and bioengineering.

[46]  D. G. Gibson,et al.  Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio natriegens. , 2018, ACS synthetic biology.

[47]  S. Kubick,et al.  Cell‐free protein expression based on extracts from CHO cells , 2014, Biotechnology and bioengineering.

[48]  C. J. Murray,et al.  Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines , 2011, Biotechnology and bioengineering.

[49]  James R. Swartz,et al.  Transforming biochemical engineering with cell-free biology , 2012 .

[50]  E. Smit,et al.  Affordable Luciferase Reporter Assay for Cell-Based High-Throughput Screening , 2013, Journal of biomolecular screening.

[51]  J. Ahn,et al.  Development of a rapid and productive cell-free protein synthesis system , 2006 .

[52]  Daniel G. Bracewell,et al.  Cell free protein synthesis: a viable option for stratified medicines manufacturing? , 2017 .

[53]  Alex J. Spice,et al.  Synthesis and Assembly of Hepatitis B Virus-Like Particles in a Pichia pastoris Cell-Free System , 2020, Frontiers in Bioengineering and Biotechnology.

[54]  Shigeyuki Yokoyama,et al.  An efficient mammalian cell-free translation system supplemented with translation factors. , 2006, Protein expression and purification.

[55]  J. Hubbuch,et al.  High‐throughput characterization of an insect cell‐free expression , 2014 .

[56]  Mathilde Koch,et al.  Large scale active-learning-guided exploration for in vitro protein production optimization , 2020, Nature Communications.

[57]  D. Raleigh,et al.  Ionic strength effects on amyloid formation by amylin are a complicated interplay among Debye screening, ion selectivity, and Hofmeister effects. , 2012, Biochemistry.

[58]  Anders Pedersen,et al.  Rational improvement of cell-free protein synthesis. , 2011, New biotechnology.

[59]  C. Anderson,et al.  Preparation of a cell-free protein-synthesizing system from wheat germ. , 1983, Methods in enzymology.

[60]  C. Pace,et al.  Toward a molecular understanding of protein solubility: increased negative surface charge correlates with increased solubility. , 2012, Biophysical journal.