The Locational Impact of Site-Specific PEGylation: Streamlined Screening with Cell-Free Protein Expression and Coarse-Grain Simulation

Abstract

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.

Engineering Cell‐Free Protein Synthesis for High‐Yield Production and Human Serum Activity Assessment of Asparaginase: Toward On‐Demand Treatment of Acute Lymphoblastic Leukemia

“…E. coli cell extract containing bacteriophage T7 RNA polymerase was prepared as previously described …”

“…25 vol% cell extract was added to 12 n m plasmid DNA and the following components: 10–20 m m magnesium glutamate (concentration optimized for protein yield), 1 m m 1,4‐diaminobutane, 1.5 m m spermidine, 40 m m phosphoenolpyruvate, 10 m m ammonium glutamate, 175 m m potassium glutamate, 2.7 m m potassium oxalate, 0.33 m m nicotinamide adenine dinucleotide, 0.27 m m coenzyme A, 1.2 m m ATP, 0.86 m m CTP, 0.86 m m GTP, 0.86 m m UTP, 0.17 m m folinic acid, and 2 m m of all the canonical amino acids except glutamic acid, and incubated at 37 °C. CFPS reactions producing crisantaspase were incubated at 30 °C shaking at 200 RPM, and selected reactions were supplemented with 2 µL 40 m m ASP every 30 min for 330 min, and a total reaction time of 7 h. Protein yield was quantified with C 14 leucine as previously described . 4 µL CFPS containing C 14 ‐labeled protein was loaded directly onto NuPAGE 4–12% bis–tris gel (Invitrogen) before electrophoresis in MES buffer (50 m m MES [2‐( N ‐morpholino)ethanesulfonic acid], 50 m m Tris base, 1 m m EDTA, 0.1% w/v SDS) at 200 V for 35 min.…”

“…4 µL CFPS containing C 14 ‐labeled protein was loaded directly onto NuPAGE 4–12% bis–tris gel (Invitrogen) before electrophoresis in MES buffer (50 m m MES [2‐( N ‐morpholino)ethanesulfonic acid], 50 m m Tris base, 1 m m EDTA, 0.1% w/v SDS) at 200 V for 35 min. The resultant gel was visualized with autoradiography as previously described . Plasmid used to express GFP and crisantaspase were reported previously…”

Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

“…The crisantaspase gene with a C‐terminal linker (GGSGGS) and 6‐His tag was purchased and cloned into the pY71 vector (GenScript). Crisantaspase CFPS reactions were performed similarly to previously described CFPS reactions, varying the yield using 12–36 nM DNA and incubating at 30 °C for 8 h or 37 °C for 3 h . Reactions were performed in 20–400 μL volumes in 2 mL microcentrifuge tubes (Bioexpress, Kaysville, UT) or 15 mL falcon tubes.…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Notably, genomic material withdrawal from chassis organism bestows circumstances in which desired synthesis reactions are achievable at high rates . Thus, such features enable prevailing utilization of CFPS systems, which are also complementary to in vivo expression for various applications such as biocatalyst development (Rolf, Rosenthal, & Lütz, 2019), complex product fabrication (Chi, Wang, Li, Ren, & Huang, 2015), disease detection (Soltani, Davis, Ford, Nelson, & Bundy, 2018), glycoprotein synthesis (Jaroentomeechai et al, 2018), prototyping minimal cells (Yue, Zhu, & Kai, 2019), synthetic gene networks (Dubuc et al, 2019), as well as protein engineering (Hong, Kwon, & Jewett, 2014;Venkat, Chen, Gan, & Fan, Sheng, Lei, Yuan and Feng (2017) Functional CRISPR gene editing toolkit Marshall et al (2018) Pathway/network prototyping UDP-N-acetylglucosamine and UDP-GlcNAc pathway Zhou et al (2010) CRISPR-mediated gene activation and repression of the reporter and endogenous genes in mammalian cells Nakamura et al (2019) Protein engineering E. coli strain mutant for release factor 1 allows turning UAG termination codon into a sense codon for site-specific incorporation of nonnatural amino acids into proteins for selective conjugation with different biomolecules Adachi et al (2019) Incorporation of uAA AzF allows for site-specific mono and dipegylation of T4 Lysozyme Wilding et al (2018) Incorporating optimized nonnatural amino acids site-specifically, the feasibility of conjugating a DBCO-PEG-monomethyl auristatin (DBCO-PEG-MMAF) drug by para-azidomethyl-L-phenylalanine (pAMF) to the tumor-specific, Her2-binding IgG Trastuzumab Zimmerman et al (2014) Biosensing Cheomogenic detection of estrogenic endocrine disruptors (hERb) in human blood and urine Salehi et al (2018) Glycoengineering Site-specific controlled glycosylation of proteins (glycoproteins) using E. coli extracts enriched for oligosaccharyltransferases and lipidlinked oligosaccharides Jaroentomeechai et al (2018) Site-directed incorporation of AzF, as well as ppropargyloxyphenylalanine from Sf21 insect cell, extracts for engineered O-glycosylation site of EPO Zemella et al (2018) Protein evolution: ribosome display Protein-tRNA-ribosome-mRNA complex for affinity selection of the nascent peptides on an immobilized monoclonal antibody specific for the peptide dynorphin Mattheakis, Bhatt and Dower (1994) Protein evolution: mRNA display Protein-puromycin-mRNA complex Roberts...…”