Enhanced protein stability through minimally invasive, direct, covalent, and site‐specific immobilization

Bioconjugating protein to nonbiological surfaces is an essential component of many promising biotechnologies impacting diverse applications such as medical diagnostics, biocatalysis, biohazard detection, and proteomics. However, to enable the widespread economical use of immobilized‐protein technologies, long‐term stability, and reusability is essential. To enhance protein stability in harsh conditions, herein we report a minimally invasive and covalent bioconjugation that enables precise control of the immobilization location at potentially any surface‐accessible location where the incorporated unnatural amino acid does not impact protein structure and function. Specifically, the PRECISE system is introduced where a uniquely reactive unnatural amino acid was incorporated site‐specifically at a prespecified location in GFP using cell‐free protein synthesis. The GFP was then directly and covalently attached to superparamagnetic beads by the unnatural amino acid in a single click reaction. The immobilized GFP was probed for retained activity and stability under harsh conditions including freeze‐thaw cycling and incubation in urea at elevated temperatures. The immobilized GFP was more stable compared to unattached protein in all cases and for all durations observed. The enhanced stability of the immobilized protein is a promising step towards long‐term protein stability for biocatalysis and other immobilized‐protein applications. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013

[1]  Bradley Charles Bundy,et al.  Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing. , 2012, BioTechniques.

[2]  Carolyn R Bertozzi,et al.  Bringing chemistry to life , 2011, Nature Methods.

[3]  Michael D. Abràmoff,et al.  Image processing with ImageJ , 2004 .

[4]  A. Kiener,et al.  Industrial biocatalysis today and tomorrow , 2001, Nature.

[5]  Jennifer A. Prescher,et al.  Chemistry in living systems , 2005, Nature chemical biology.

[6]  Alexander Sczyrba,et al.  High-throughput in vitro glycoside hydrolase (HIGH) screening for enzyme discovery. , 2011, Angewandte Chemie.

[7]  M. Goto,et al.  Exploring enzymatic catalysis at a solid surface: a case study with transglutaminase-mediated protein immobilization. , 2007, Organic & biomolecular chemistry.

[8]  C. Poulter,et al.  Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids. , 2006, Journal of the American Chemical Society.

[9]  P. Schultz,et al.  An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity. , 2011, Biochemistry.

[10]  B. Hingerty,et al.  Further refinement of the structure of yeast tRNAPhe. , 1978, Journal of molecular biology.

[11]  Renjie Liao,et al.  Properties of Rhodotorula gracilis D-amino acid oxidase immobilized on magnetic beads through his-tag. , 2008, Journal of bioscience and bioengineering.

[12]  H. Schoemaker,et al.  Dispelling the Myths--Biocatalysis in Industrial Synthesis , 2003, Science.

[13]  Roger A. Sheldon,et al.  Enzyme Immobilization: The Quest for Optimum Performance , 2007 .

[14]  J. Feijen,et al.  Protein immobilization strategies for protein biochips. , 2007, Biomacromolecules.

[15]  Duane E. Prasuhn,et al.  Unnatural amino acid incorporation into virus-like particles. , 2008, Bioconjugate chemistry.

[16]  James R. Swartz,et al.  High‐level cell‐free synthesis yields of proteins containing site‐specific non‐natural amino acids , 2009, Biotechnology and bioengineering.

[17]  Ki-Chul Jung,et al.  Fluorescence Quenching of Green Fluorescent Protein during Denaturation by Guanidine , 2005 .

[18]  Abraham Ulman,et al.  Activity of Candida rugosa lipase immobilized on gamma-Fe2O3 magnetic nanoparticles. , 2003, Journal of the American Chemical Society.

[19]  Sajja Hari Krishna,et al.  Strategies for Improving Enzymes for Efficient Biocatalysis , 2004 .

[20]  Kim A Woodrow,et al.  Cell-free protein synthesis with prokaryotic combined transcription-translation. , 2004, Methods in molecular biology.

[21]  Túlio Marcos Santos,et al.  Up-To-Date Insight on Industrial Enzymes Applications and Global Market , 2012 .

[22]  Xiaoning Jing,et al.  PEGylation of bovine serum albumin using click chemistry for the application as drug carriers , 2012, Biotechnology progress (Print).

[23]  M. Finn,et al.  Tailored ligand acceleration of the Cu-catalyzed azide-alkyne cycloaddition reaction: practical and mechanistic implications. , 2010, Journal of the American Chemical Society.

[24]  Hee-Sung Park,et al.  Controlled and oriented immobilization of protein by site-specific incorporation of unnatural amino acid. , 2011, Analytical chemistry.

[25]  Kedar G. Patel,et al.  Surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry. , 2011, Bioconjugate chemistry.

[26]  Ramesh N. Patel,et al.  Biocatalytic synthesis of intermediates for the synthesis of chiral drug substances. , 2001, Current opinion in biotechnology.

[27]  Bradley C. Bundy,et al.  Efficient disulfide bond formation in virus-like particles. , 2011, Journal of biotechnology.

[28]  Peter G Schultz,et al.  Unnatural amino acid mutagenesis of green fluorescent protein. , 2003, The Journal of organic chemistry.

[29]  James R. Swartz,et al.  Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation. , 2010, Bioconjugate chemistry.

[30]  M. Finn,et al.  "Clickable" agarose for affinity chromatography. , 2005, Bioconjugate chemistry.

[31]  James H. Clark,et al.  Chemistry goes green. , 2009, Nature chemistry.

[32]  L. DeLucas,et al.  His-tag impact on structure. , 2007, Acta crystallographica. Section D, Biological crystallography.

[33]  P G Schultz,et al.  Expanding the Genetic Code of Escherichia coli , 2001, Science.

[34]  Peter G Schultz,et al.  Adding new chemistries to the genetic code. , 2010, Annual review of biochemistry.

[35]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[36]  Richard J. Fox,et al.  Principles of Enzyme Optimization for the Rapid Creation of Industrial Biocatalysts , 2011 .

[37]  John A Tainer,et al.  Structural chemistry of a green fluorescent protein Zn biosensor. , 2002, Journal of the American Chemical Society.

[38]  E.E. Pissaloux,et al.  Image Processing , 1994, Proceedings. Second Euromicro Workshop on Parallel and Distributed Processing.

[39]  N. Stephanopoulos,et al.  Choosing an effective protein bioconjugation strategy. , 2011, Nature chemical biology.

[40]  Charlotte K. Williams,et al.  The Path Forward for Biofuels and Biomaterials , 2006, Science.

[41]  Daniel I. C. Wang,et al.  Recyclable nanobiocatalyst for enantioselective sulfoxidation: facile fabrication and high performance of chloroperoxidase-coated magnetic nanoparticles with iron oxide core and polymer shell. , 2009, Journal of the American Chemical Society.

[42]  Peter G Schultz,et al.  An enhanced system for unnatural amino acid mutagenesis in E. coli. , 2010, Journal of molecular biology.

[43]  Dawei Zhang,et al.  Site-specific fluorescent labeling of nascent proteins on the translating ribosome. , 2011, Journal of the American Chemical Society.

[44]  T. Delair,et al.  Covalent immobilization of proteins onto (Maleic anhydride-alt-methyl vinyl ether) copolymers: enhanced immobilization of recombinant proteins. , 1998, Bioconjugate chemistry.

[45]  Peter G. Schultz,et al.  A chemical toolkit for proteins — an expanded genetic code , 2006, Nature Reviews Molecular Cell Biology.

[46]  T. Terwilliger,et al.  Engineering and characterization of a superfolder green fluorescent protein , 2006, Nature Biotechnology.

[47]  M. Sisido,et al.  Incorporation of nonnatural amino acids into proteins by using various four-base codons in an Escherichia coli in vitro translation system. , 2001, Biochemistry.

[48]  Bradley C. Bundy,et al.  The incorporation of the A2 protein to produce novel Qβ virus‐like particles using cell‐free protein synthesis , 2012, Biotechnology progress.

[49]  C. Schmidt-Dannert,et al.  Directed Enzyme and Pathway Evolution , 2011 .

[50]  Manuel Ferrer,et al.  Environmental biocatalysis: from remediation with enzymes to novel green processes. , 2006, Trends in biotechnology.

[51]  Uwe T Bornscheuer,et al.  Immobilizing enzymes: how to create more suitable biocatalysts. , 2003, Angewandte Chemie.

[52]  M. Finn,et al.  Analysis and optimization of copper-catalyzed azide-alkyne cycloaddition for bioconjugation. , 2009, Angewandte Chemie.

[53]  Bradley C. Bundy,et al.  Escherichia coli‐based cell‐free synthesis of virus‐like particles , 2008, Biotechnology and bioengineering.

[54]  C. Niemeyer.  The developments of semisynthetic DNA-protein conjugates. , 2002, Trends in biotechnology.