Evidence map›Paper›PMID 42026235›Full record

ArticleMolecular systems biology2026

Fast analysis and engineering of protein function by microbe-independent deep assembly and screening.

Yan Wu, Pengli Wang, Lan Xiang Liu, Daesun Song, Qin Qin, Chao Gao, Matt Hageman, Thomas A Kirkland, Yichi Su, Michael Z Lin

Erratum issuedAbstract read
PubMed Full text at PMC Publisher
In one paragraph

Article in Molecular systems biology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. An erratum has been issued. Cited by 1 paper.

0numbers the graph read from it
0cells of the map it votes in
1citing papers in PubMed
–field-weighted citation impact
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

1 citing paper in PubMed.

  1. Article
4 · The record

Corrections and comments

5 · Who and what money

Authors and funding

10 authors.

Yan Wu *Department of Bioengineering, Stanford University, Stanford, CA, USA.
Pengli Wang *Department of Chemical Engineering, Stanford University, Stanford, CA, USA.ORCID http://orcid.org/0000-0002-6368-0627
Lan Xiang LiuDepartment of Neurobiology, Stanford University, Stanford, CA, USA.
Daesun SongDepartment of Neurobiology, Stanford University, Stanford, CA, USA.ORCID http://orcid.org/0000-0002-6206-2034
Qin QinInstitute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China.
Chao GaoPromega Corporation, San Luis Obispo, CA, USA.ORCID http://orcid.org/0000-0003-3662-6518
Matt HagemanPromega Corporation, San Luis Obispo, CA, USA.
Thomas A KirklandPromega Corporation, Madison, WI, USA.ORCID http://orcid.org/0000-0003-0859-5785
Yichi SuInstitute for Translational Brain Research, State Key Laboratory of Medical Neurobiology, MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China.ORCID http://orcid.org/0000-0001-5715-835X
Michael Z LinDepartment of Bioengineering, Stanford University, Stanford, CA, USA. mzlin@stanford.edu.ORCID http://orcid.org/0000-0002-0492-1961

Funding

Noninvasive bioluminescent imaging of neuronal activity in freely behaving animalsR21DA048252 · NIDA · STANFORD UNIVERSITY · PI LIN, MICHAEL Z. · 2019 to 2020
$435k
Bioluminescent indicators for noninvasive imaging of acetylcholine releaseR21NS122055 · NINDS · STANFORD UNIVERSITY · PI LIN, MICHAEL Z. · 2021 to 2021
$434k
HHS | NIH | National Institute of Neurological Disorders and Stroke (NINDS) 1R21NS122055HHS | NIH | National Institute on Drug Abuse (NIDA) 1R21DA048252NIDA NIH HHS R21 DA048252NINDS NIH HHS R21 NS122055
6 · The paper itself

Abstract

Traditional methods for engineering and sequence-fitness analysis of proteins in mammalian cells are limited by the time, cost, and labor associated with plasmid cloning and preparation. Here we present Microbe-Independent Deep Assembly and Screening (MIDAS), a deterministic platform for rapid protein variant expression and characterization in mammalian cells that bypasses microbial cloning by directly transfecting PCR-assembled genes. MIDAS enables high-quality sequence-fitness assessment of arbitrary mutational spaces, including truly deep saturation mutagenesis and combinatorial variant assembly, requiring less than one workday from initial PCR to cell transfection. Using MIDAS, we engineer a high-performance acetylcholine neurotransmitter bioluminescent indicator (ACh-NeuBI), achieving stepwise improvements in responsivity through linker engineering, single-site, and multi-site mutagenesis. We also apply MIDAS to engineer improved NanoLuc luciferase variants for multiple substrates, and to characterize the structural basis of mutational tolerance and substrate specificity. Thus, MIDAS is a versatile method for rapid plasmid-free protein engineering and sequence-fitness analysis in mammalian cells, offering a practical alternative to cloning-based approaches for many protein optimization and characterization tasks.

Indexed as

Protein EngineeringAnimalsCloning, MolecularHEK293 CellsHumansLuciferasesMutagenesisMutagenesis, Site-DirectedPlasmidsTransfectionLuciferases

Identifiers

PMID42026235
PMCPMC13230610

What OpenQuestion holds

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Registered trials

None linked

Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.