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bioRxiv2024

Characterisation of RNA guanine-7 methyltransferase (RNMT) using a small molecule approach

The maturation of the RNA cap involving guanosine N-7 methylation, catalyzed by the HsRNMT (RNA guanine-7 methyltransferase)-RAM (RNA guanine-N7 methyltransferase activating subunit) complex, is currently under investigation as a novel strategy to combat PIK3CA mutant breast cancer. However, the development of effective drugs is hindered by a limited understanding of the enzymes mechanism and a lack of small molecule inhibitors. Following the elucidation of the HsRNMT-RAM molecular mechanism, we report the biophysical characterization of two small molecule hits. Biophysics, biochemistry and structural biology confirm that both compounds bind competitively with cap and bind effectively to HsRNMT-RAM in the presence of the co-product SAM, with a binding affinity (KD) of approximately 1 M. This stabilisation of the enzyme-product complex results in uncompetitive inhibition. Finally, we describe the properties of the cap pocket and provided suggestions for further development of the tool compounds.

biochemistry↗

bioRxiv2024

Structural and molecular basis of FAN1 defects in promoting Huntington's disease

FAN1 is a DNA dependent nuclease whose proper function is essential for maintaining human health. For example, a genetic variant in FAN1, Arg507 to His hastens onset of Huntingtons disease, a repeat expansion disorder for which there is no cure. How the Arg507His mutation affects FAN1 structure and enzymatic function is unknown. Using cryo-EM and biochemistry, we have discovered that FAN1 arginine 507 is critical for its interaction with PCNA, and mutation of Arg507 to His attenuates assembly of the FAN1-PCNA on a disease-relevant extrahelical DNA extrusions formed within DNA repeats. This mutation concomitantly abolishes PCNA-FAN1-dependent cleavage of such extrusions, underscoring the importance of PCNA to the genome stabilizing function of FAN1. These results unravel the molecular basis for a specific mutation in FAN1 that dramatically hastens the onset of Huntingtons disease.

biochemistry↗

bioRxiv2024

PSKH1 kinase activity is differentially modulated via allosteric binding of Ca2+ sensor proteins

Protein Serine Kinase H1 (PSKH1) was recently identified as a crucial factor in kidney development and is overexpressed in prostate, lung and kidney cancers. However, little is known about PSKH1 regulatory mechanisms, leading to its classification as a "dark" kinase. Here, we used biochemistry and mass spectrometry to define PSKH1s consensus substrate motif, protein interactors, and how interactors, including Ca2+ sensor proteins, promote or suppress activity. Intriguingly, despite the absence of a canonical Calmodulin binding motif, Ca2+-Calmodulin activated PSKH1 while, in contrast, the ER-resident Ca2+ sensor of the CREC family, Reticulocalbin-3, suppressed PSKH1 catalytic activity. In addition to antagonistic regulation of the PSKH1 kinase domain by Ca2+ sensing proteins, we identified UNC119B as a protein interactor that activates PSKH1 via direct engagement of the kinase domain. Our findings identify complementary allosteric mechanisms by which regulatory proteins tune PSKH1s catalytic activity, and raise the possibility that different Ca2+ sensors may act more broadly to tune kinase activities by detecting and decoding extremes of intracellular Ca2+ concentrations.

biochemistry↗

bioRxiv2024

Getting Started with Machine Learning for Experimental Biochemists and Other Molecular Scientists

Machine learning (ML) is rapidly gaining traction in many areas of experimental molecular science for elucidating relationships and patterns in large or complex data sets. Historically, ML was largely the preserve of those with specialized training in fields such as statistics or cheminformatics. Increasingly, however, ML methodologies are becoming part of the standard toolkit for experimental scientists across a range of disciplines. Lowering the barrier of entry to these ML techniques, for scientists without a significant background in computer science or statistics, is important to broadening access to these powerful methods. Here we provide detailed, step by step tutorials for performing four ML methods that are particularly useful for applications in biochemistry, cell biology, and drug discovery: hierarchical clustering, Principal Component Analysis (PCA), Partial Least-Squares Discriminant Analysis (PLSDA), and Partial Least-Squares Regression (PLSR). The protocols are written for the widely used software MATLAB, but no prior experience with MATLAB is required to use them. We include an explanation of each step, pitched at a level to be understood by investigators without any prior experience with ML, MATLAB, or any kind of coding. We also highlight the scientific issues pertaining to selecting and scaling the data to be analyzed, and describe controls to test the validity of the results obtained. Throughout, we emphasize the relationship between the scientific question and how to choose data and methods that will allow it to be addressed in a meaningful way. Our aim is to provide a basic introduction that will equip experimental chemical biologists and other chemical and biomedical scientists with the knowledge required to use ML to aid in the design of experiments, the formulation and data-driven testing of hypotheses, and the analysis of experimental data. Basic Protocol 1Clustering Basic Protocol 2Principal Component Analysis (PCA) Basic Protocol 3Partial Least Squares Discriminant Analysis (PLSDA) Basic Protocol 4Partial Least Squares Regression (PLSR)

biochemistry↗

bioRxiv2024

Structural basis of pseudoGTPase-mediated protein-protein interactions

GTPases regulate various cellular processes through conformational changes triggered by GTP or GDP binding. Recently, pseudoGTPases, the catalytically inactive counterparts of GTPases, have been identified across species from bacteria to human, although their functions and mechanisms remain unexplored. In this study, we demonstrate that the N-terminal region of the assembly chaperone AAGAB is a type i pseudoGTPase using biochemistry and X-ray crystallography. Furthermore, we discovered that the AAGAB pseudoGTPase domain (psGD) interacts with the {sigma} subunits of AP1 and AP2 adaptor complexes, heterotetrameric complexes involved in clathrin-mediated membrane trafficking. AAGAB psGD engages the {sigma} subunits via a unique interface distinct from the conventional GTPase interacting regions. Further biochemical and cell-based assays confirmed the crucial role of the newly identified interface in binding and membrane trafficking. Collectively, our results establish AAGAB pseudoGTPase domain as a critical protein-protein interaction module. These findings offer new insight into the structural basis and molecular mechanisms of pseudoGTPases. HighlightsO_LIAAGAB N-terminal region is a pseudoGTPase C_LIO_LIAAGAB pseudoGTPase domain interacts with and stabilizes AP1 and AP2 {sigma} subunits C_LIO_LIThe crystal structure of AAGAB psGD:AP1{sigma}3 is the first reported psGD complex structure, revealing a unique interface independent of guanine nucleotide regulation C_LIO_LIThe psGD:AP1{sigma}3 structure offers mechanistic insights into {sigma} subunits stabilization and protection through adaptor complex assembly C_LI

biochemistry↗

bioRxiv2024

Biochemical, structural, and single-molecule characterization of LIG1 active site mutants demonstrate role of F635 and F872 residues for faithful ligation

Human DNA ligase 1 (LIG1) finalizes DNA repair pathways by an ultimate ligation step and discriminates against nicks containing unusual ends, yet the contribution of the conserved active site residues for faithful end joining remains unknown. Here, using biochemistry, X-ray crystallography, and single-molecule approaches, we comprehensively characterized LIG1 mutants carrying Ala(A) and Leu(L) substitutions at the active site residues Phe(F)635 and Phe(F)872. Our results showed an abolished ligation of nick DNA substrates with all 12 non-canonical mismatches, while the mutagenic nick sealing of oxidatively damaged ends by wild-type enzyme is significantly reduced by F635A/L and F872A/L substitutions. Furthermore, sugar discrimination against a single ribonucleotide at 3- or 5-end of nick DNA is distinctly affected depending on architecture of 3-terminus:template base pairing. Finally, our LIG1 structures demonstrated the importance of DNA end alignment governed by the distance to nick site through F635 and F872 residues, and single-molecule measurements showed similar nick DNA binding modes for LIG1 wild-type and active site mutants in real-time. Overall, our study provides a mechanistic insight into the mechanism by which conserved F635 and F872 residues contribute to ligation efficiency of nick repair intermediates that mimic DNA polymerase-mediated mismatch, damaged, or ribonucleotide insertion products and how LIG1 ensures faithful end joining at the final step of DNA repair to maintain genome integrity.

biochemistry↗

bioRxiv2025

A suite of pre-assembled, pET28b-based Golden Gate vectors for efficient protein engineering and expression

Expression and purification of recombinant proteins in E. coli is a bedrock technique in biochemistry and molecular biology. Expression optimization requires testing different combinations of solubility tags, affinity purification techniques, and site-specific proteases. This optimization is laborious and time consuming as these features are spread across different vector series and require different cloning strategies with varying efficiencies. Modular cloning kits based on the Golden Gate system exist, but are overly complicated for many applications, such as undergraduate research or simple screening of protein purification features. An ideal solution is for a single gene synthesis or PCR product to be compatible with a large series of pre-assembled Golden Gate vectors containing a broad array of purification features at either the N or C-terminus. To our knowledge, no such system exists. To fulfill this unmet need, we Golden Gate domesticated the pET28b vector and developed a suite of 21 vectors with different combinations of purification tags, solubility domains, visualization/labeling tags, and protease sites. We also developed a completely scarless vector series with 9 different N-terminal tags. The system is modular, allowing users to easily customize the vectors with their preferred combinations of features. To allow for easy visual screening of cloned vectors, we optimized constitutive expression of the fluorescent protein mScarlet3 in the reverse strand, resulting in a red to white color change upon successful cloning. Testing with the model protein sfGFP shows the ease of visual screening, high efficiency of cloning, and robust protein expression. These vectors provide versatile, high-throughput solutions for protein engineering and functional studies in E. coli.

biochemistry↗

bioRxiv2025

ST-FFPE-mIF: Integrating Spatial Transcriptomics and Multiplex Immunofluorescence in Formalin-Fixed Paraffin-Embedded Tissues Using Stereo-seq

Stereo-seq is the state-of-the-art technique, which allows capturing of spatial transcriptomic data at sub-cellular resolution and in large scale of view. Stereo-seq FFPE technique is recently launched with the capability to obtain spatially resolved gene expression in FFPE tissue sections. However, the omic information is limited to transcriptome. To delineate spatial molecular hierarchy from transcriptome to phenome, we present the protocol that implements nine-plex immunofluorescence staining with detection of spatial transcriptomics in one FFPE tissue section (short for ST-FFPE-mIF). This protocol details the modified biochemistry procedure and also provides analytical framework to achieve single-cell resolution through nuclear or membrane staining-based cell segmentation. We demonstrated the application of ST-FFPE-mIF in colorectal cancer sample to illustrate how ST-FFPE-mIF facilitates a robust analysis of spatially resolved multi-omic data at the single-cell level.

biochemistry↗

bioRxiv2025

Catalytic Mechanism and Differential Alarmone Regulation of a Conserved Stringent Nucleosidase

Understanding how bacteria rapidly adapt their metabolism in response to external stimuli is key to addressing the present crisis of antibiotics-resistant infections. In the Gram-negative bacterium Escherichia coli, the universal stringent response is elicited in response to some antibiotics and involves production of the global alarmones, (p)ppGpp, which bind directly to many cellular targets. The nucleosidase PpnN that cleaves nucleotides into 5-phosphate ribose and nucleobase, was shown to be a target of (p)ppGpp and control the delicate balance of bacterial fitness and persistence to fluoroquinolone antibiotics, thus conferring optimal survival strategies to bacteria during antibiotic selective pressure. Although both pppGpp and ppGpp stimulate the enzymatic activity of PpnN, they exert distinct effects on the enzymes cooperativity. The molecular mechanism underlying this subtle difference as well as the precise catalytic mechanism of PpnN, remain obscure. In this study, we provide mechanistic insights into the interaction of PpnN with substrate analogue, reaction products and alarmone molecules, which allows us to understand the catalytic mechanism of this family of nucleosidases and the differential modes of regulation by ppGpp and pppGpp, respectively. Comparison with the homologous LOG proteins involved in cytokinin production in plants reveals an ancient, universal mechanism for cleaving purine monophosphates that bacteria have incorporated regulatory controls through alarmones upon stringent responses. IMPORTANCEThis study explores the distinct manners that the stringent alarmones pppGpp and ppGpp interact with the nucleosidase PpnN, which diverge from their typical, consistent effects on other target proteins. Furthermore, PpnN plays a key role in balancing bacterial fitness and tolerance to antibiotics, yet its catalytic mechanism has remained unclear. Through a combination of structural biology, molecular simulations, biochemistry, mutagenesis, and physiological analyses, this research uncovers the mechanistic differences in how both alarmones differentially impact enzyme cooperativity of PpnN and activation under stress conditions. Additionally, by analysing structures of PpnN complexed with substrate analogues, reaction products, and alarmones, as well as conducting bioinformatic comparisons, we propose a conserved catalytic mechanism shared with its homologue, LOG, a protein involved in cytokinin signalling - a critical growth hormone in plants. These insights underscore the nuanced regulatory roles of alarmones and PpnN/LOG group of nucleosidases, highlighting their evolutionary diversification to meet the varied environmental survival needs across organisms.

biochemistry↗

bioRxiv2025

Characterization of nick binding and sealing by LIG1 Huntington's disease-asssociated K845N variant at biochemical, structural, and single-molecule levels

DNA ligase 1 (LIG1) joins broken strand breaks and discriminates against nicks containing mismatch or oxidative damage. Huntingtons disease (HD)-associated mutation K845N in LIG1 gene has been predicted to be onset delaying and suppresses CAG repeat expansion. Yet, how this mutation impacts faithful nick sealing and efficient DNA binding by LIG1 remains unknown. Here, using biochemistry, X-ray crystallography, and total internal reflection fluorescence microscopy, we comprehensively characterized the impact of LIG1 HD-associated mutation at biochemical, structural, and single-molecule levels. Our results showed a reduced ligation efficiency by LIG1 K845N variant in the presence of nick substrates containing all possible 12 mismatches, 8-oxoG, and ribonucleotides at the 3-end when compared with the wild-type enzyme. Furthermore, our structures provided an atomic insight into differences in distances between the functional groups of K/N845 and DNA ends, demonstrating similar conformation and a lack of large scale alternations at the ligase active site. Finally, our single-molecule measurements in real-time revealed that K845N mutant binds less frequently for shorter life-time to nick DNA than wild-type protein. Overall findings contribute to understand the mechanism by which LIG1 ensures fidelity and nick binding to maintain genome stability at the final ligation step in normal versus disease states.

biochemistry↗

bioRxiv2025

Constructing a consensus serum metabolome

Blood analysis is the most common in biomedical applications and a reference metabolome will be critical for effective annotation and for guiding scientific investigations. However, compiling such a reference is hindered by many technical challenges, despite the availability of large amount of metabolomics data today. Based on a new set of data structures and tools, we have assembled a consensus serum metabolome (CSM) from over 100,000 mass spectrometry acquisitions of more than 200 million spectra. This provides a comprehensive survey of human blood chemistry, revealing the frequency dependent nature of metabolome and exposome. Major gaps are found between CSM and the current databases. The CSM enables community-level data alignment and significantly improves annotation quality of LC-MS metabolomics. HighlightsO_LIA reference of human biochemistry linked to observation frequency C_LIO_LIMajor gaps revealed in current databases and experimental methods C_LIO_LIEnabling cross-laboratory, cross-platform data alignment C_LIO_LIAccelerated and cumulative metabolite annotation C_LI

biochemistry↗

bioRxiv2025

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

biochemistry↗

bioRxiv2025

RAD51AP1 is a versatile RAD51 modulator

RAD51AP1 is an emergent key factor in homologous recombination (HR), the major pathway for accurate repair of DNA double-strand breaks, and in alternative lengthening of telomeres (ALT). Depletion of RAD51AP1 diminishes HR and overexpression is common in cancer, where it is associated with malignancy. Here, we show that RAD51AP1 serves as a versatile modulator of the RAD51 recombinase, the central player in HR. Through a combination of biochemistry and structural biology, we reveal that RAD51AP1 possesses at least three RAD51-binding sites that facilitate its binding across two adjacent RAD51 molecules. We uncover a novel RAD51 binding mode and characterise a hitherto overlooked role for RAD51AP1 in stabilising RAD51-ssDNA filaments and promoting strand exchange. Further, we resolved structures of RAD51-ssDNA filaments in the presence of Mg2+-ATP and Mg2+-ADP, revealing conformational changes upon ATP hydrolysis and explaining how ADP reduces RAD51-DNA binding. Our findings provide mechanistic insights into RAD51 recombinase and RAD51AP1. HighlightsO_LIStructures of RAD51 filaments in the presence of Mg2+-ATP and Mg2+-ADP reveal how ATP hydrolysis reduces DNA binding C_LIO_LIATP hydrolysis does not induce RAD51 filament contraction, instead it induces filament relaxation C_LIO_LIRAD51AP1 uses three sites to bind across two RAD51 monomers C_LIO_LIRAD51AP1 uses a unique binding mode to stabilise the RAD51 N-terminal domain and protomer interface of RAD51 C_LIO_LIRAD51AP1 binding induces conformational changes that promote RAD51 DNA association, oligomerisation, filament nucleation, filament stabilisation and strand exchange C_LI

biochemistry↗

bioRxiv2025

Disease-causing mutations in the G protein β5 β-propeller disrupt its chaperonin-mediated folding trajectory

The Chaperonin Containing Tailless polypeptide 1 (CCT or TRiC) is an essential cytosolic chaperone that folds multiple protein substrates, including many with {beta}-propeller folds. One {beta}-propeller substrate is the G protein {beta}5 subunit (G{beta}5) of Regulator of G protein Signaling (RGS) complexes that determine the duration of G protein signals in neurons. In recent work, we used cryo-electron microscopy (cryo-EM) to visualize the complete CCT-mediated folding trajectory for G{beta}5, from an initiating electrostatic interaction of a single {beta}-strand in G{beta}5 with the CCT5 subunit to a completely folded {beta}-propeller structure. Here, we employed biochemistry and cryo-EM to determine key interactions with CCT that initiate G{beta}5 folding and how missense mutations in G{beta}5 that cause severe neurological diseases alter the G{beta}5 folding trajectory and lead to incompletely folded, trapped intermediates. These findings highlight how CCT recognizes folding substrates, how defects in chaperonin-mediated folding contribute to disease, and how strategies might be designed to stabilize misfolded proteins to restore function. SignificanceElectrostatic interactions between the CCT chaperonin and its protein substrates initiate the folding process. Using cryo-EM structure determinations, we found a striking specificity for these interactions that allow CCT-mediated G{beta}5 folding. Moreover, certain missense mutations in G{beta}5 lead to misfolding and are associated with neurological disorders. We tracked how these mutations disrupt the normal folding of G{beta}5 by CCT. Although mutant G{beta}5 still binds the complex, folding stalls mid-process, leaving the protein trapped in partially folded, non-functional states. These defects arise from disrupted packing of the G{beta}5 core that interferes with formation of the native structure. Our findings reveal a molecular basis for G{beta}5 misfolding in disease and suggest pharmacological chaperones that stabilize the folded state might restore proper function.

biochemistry↗

bioRxiv2025

The BRD4-nucleosome interaction is enhanced modestly and non-selectively by histone acetylation

BRD4 regulates gene transcription in complex eukaryotes, in part through the binding of its tandem bromodomains to acetylated lysine residues found in histones and transcription factors. Despite pharmacological inhibition of these domains showing promise in preclinical studies, clinical trial data have been less encouraging so far. A stronger understanding of BRD4 biochemistry could provide a route to better outcomes. To advance on prior work, which has focused almost entirely on the binding of isolated bromodomains and acetylated peptides, we have sought the preferred nucleosomal binding partner of full-length BRD4. We demonstrate that BRD4 binds with sub-micromolar affinity to both unmodified nucleosomes and to DNA alone. In strong contrast to BRD4-peptide interactions, we also find that the affinity of BRD4 for nucleosomes is increased only 2-4-fold by histone acetylation and that this affinity has little dependence on the acetylation pattern. Despite this modest effect of acetylation, binding of BRD4 to acetyllysine in the nucleosome was more resistant to perturbation by mutation or small-molecule inhibition than BRD4-peptide interactions. Our work helps bridge the gap between cellular and prior in vitro work and provides clues to explain the in vivo chromatin occupancy profile of BRD4 and how it changes upon therapeutic inhibition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/656505v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1aa532aorg.highwire.dtl.DTLVardef@13a154forg.highwire.dtl.DTLVardef@4d2039org.highwire.dtl.DTLVardef@17e8377_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Purification of post-transcriptionally modified tRNAs for enhanced cell-free translation systems

Transfer RNAs (tRNAs) are utilized by the ribosome to decode the nucleic acid alphabet. tRNA structure, stability, aminoacylation efficiency, and decoding efficacy are governed by their extensive post-transcriptional modifications. In most studies, individual tRNAs are generated using in vitro transcription, which produces tRNAs devoid of these critical site-specific modifications, negatively affecting translation yields and fidelity. To address this, we have developed a purification method which couples tRNA overexpression to DNA hybridization-based purification. Using this approach, we produced native tRNAs from E. coli in high yield and purity while retaining their complement of native post-transcriptional modifications and translational activity. We extend this technique to the purification of [Formula] and [Formula], tRNAs of critical importance for genetic code expansion. We confirmed that both [Formula] and [Formula] contain native E. coli post-transcriptional modifications and provide the first complete modification profiles of each. Moreover, we found that in vivo-generated [Formula] significantly outperforms its in vitro-generated counterpart in amber codon suppression in cell-free translation reactions. Finally, we purified an engineered variant of E. coli [Formula], extending our studies to synthetic tRNAs. We present a flexible method which generates modified tRNAs in high yield and purity, addressing a critical and persistent challenge in RNA biochemistry. This toolkit enables future structural and cell-free studies through scalable access to native and engineered tRNAs, advancing the broader field of translation and synthetic biology.

biochemistry↗

bioRxiv2025

Convergent Functional Constraints Make Biochemical Reaction Diversity Predictable

The predictability of biochemical evolution remains highly debated, as it is unknown if biological function can be predicted a priori. Here, we explore predictability of function not in individual reactions, but in ensembles. Sampling from 13,777 bacterial, 371 archaeal, and 203 eukaryotic genomes, we use Enzyme Commission (EC) classification to hierarchically group enzyme-catalyzed reactions that perform similar transformations into functional equivalence classes. We show that organisms partition their reaction diversity among these functional equivalence classes in a predictable way, identifying over 120 new, system-size dependent functional scaling relationships. We find distantly related lineages, acting on distinct molecular substrates, can display similar functional scaling, demonstrating that convergence is not driven by phylogeny or common reactions. We demonstrate how transitions in functional scaling can be identified with physiological shifts, using as case studies O2-utilizing oxidoreductases and hexosyltransferases. Taken together, our results open novel avenues for predicting global features of evolving enzyme populations, independent of protein structures. These functional constraints may have broad implications, including for predicting biochemical diversity, designing synthetic organisms, and modeling the evolution of reaction diversity in cases where the exact identity of catalysts is not known, such as during the emergence of life and for potential alternative forms of biochemistry.

biochemistry↗

bioRxiv2025

An Automated End-to-End Workflow for Production of Secreted Proteins in Transfected Mammalian Cells

The advancement of automation technologies has helped to enable a surge in large-scale screening efforts across fields such as molecular biology, protein biochemistry, cell biology, and structural biology. In the context of this "omics"-driven research, there is a need to generate automation platforms that are more flexible and less expensive, so that they can be utilized for basic research conducted by small groups. A key challenge in automation lies in developing methods that can replicate fine motor techniques that are normally performed manually by researchers at the bench. We are engaged in a large-scale project to map interactions among human cell-surface and secreted proteins and assess their effects on cells. This project involves production of a library of more than 2000 recombinant His-tagged fusion proteins secreted from transfected Expi293 cells. To execute such a project with a small group at an academic institution required construction of an affordable automated system that could also be used by other investigators. This led us to develop a high-throughput, 96-well format automation platform for end-to-end protein production. The workflow includes transformation of E. coli, plasmid DNA preparation, transient transfection, protein purification, desalting and buffer exchange, protein quantification, and normalization of protein concentrations, resulting in assay-ready proteins. The system is built around an in-house engineered modular robotic platform that integrates liquid handling with a suite of interchangeable plug-and-play mobile enclosed device modules. Housed within a BSL-2 sterile environment, the platform enables flexible, fully automated workflows and can be readily customized for diverse user-defined protocols.

biochemistry↗