Library · Biology & lab methods
What Transgenic & Recombinant Expression got wrong, from 33 theses
Summary written by a model from the records below: Recombinant expression systems frequently fail due to target protein aggregation into insoluble inclusion bodies, complete absence of protein yields, and expression-induced host toxicity. Downstream recovery is further hindered by persistent co-purification of host contaminants, absent post-translational modifications, and defective structural assembly. These records come from graduate theses, 2021 to 2026, each labeled with its degree where the repository states it, and each links to its thesis. They were extracted by language models reading the full text, so treat each as a lead to read, not a verdict.
Plus 9 problems the authors left open, listed at the end.
Failure modes, by theses reporting them
Select a mode to jump to its records.
Recombinant proteins form insoluble aggregates and inclusion bodies during expression
Target proteins frequently partitioned into insoluble inclusion bodies or suffered irreversible aggregation across bacterial and insect expression systems despite the use of solubility tags, truncations, or altered incubation conditions. These aggregation events prevented the recovery of soluble native targets and caused failure in downstream affinity binding.
Tried and failed
native recombinant protein expression with solubility tags applied to truncated plant protein domains. Reason: expressed proteins formed insoluble inclusion bodies rather than soluble native protein
Quantitative characterization of the interactions between LRT2, Hsp90, and OsIAA11 that regulate the development of lateral roots in rice plants · Cornell · PhD
Tried and failed
recombinant bacterial expression with affinity chromatography applied to bacterial outer membrane proteins. Reason: protein aggregated into insoluble inclusion bodies and failed affinity column binding
The interaction of glycosylated major outer membrane protein of Campylobacter jejuni with natural and synthetic ligands · Nottingham · PhD
Tried and failed
low-temperature low-inducer bacterial expression applied to insoluble recombinant multi-domain human proteins. Reason: protein remained completely partitioned into insoluble inclusion bodies despite reduced expression kinetics
Establishing The Foundations For Biophysical Studies Of Allostery In Parkin E3 Ubiquitin Ligase · Penn · PhD
Tried and failed
vesicle nucleating peptide tagging for protein secretion applied to recombinant fusion protein expression. Reason: failed to export vesicles and resulted exclusively in insoluble protein aggregation
Tried and failed
recombinant protein expression and solubilization applied to heterologous full-length eukaryotic protein. Reason: insoluble protein aggregation in bacterial pellets
Investigating the Role of Drosophila Tomosyn in Synaptic Strength and Plasticity · MIT · PhD
Tried and failed
recombinant protein expression in bacterial and insect hosts applied to full-length multidomain E3 ligases. Reason: constructs formed insoluble aggregates in both bacterial and insect cell expression systems
Structural and functional characterisation of Class IV TRIM E3 ligases · Imperial · Master's
Lost to a baseline
NoCAR recombinant expression in OMW produced 146.24 µg/mL insoluble inclusion bodies (122.5% higher than shake flasks at 65.73 µg/mL) and 19.29% lower soluble yield, attributed to temperature gradients during shaking incubation.
Validierung von 3D-gedruckten Reaktorsystemen für biotechnologische Applikationen · Leibniz Universität Hannover · PhD
Considered and rejected
Considered and rejected: Rejected recombinant expression of MOMP in E. coli due to inclusion body formation and poor His-tag binding, opting for native extraction directly from C. jejuni.
The interaction of glycosylated major outer membrane protein of Campylobacter jejuni with natural and synthetic ligands · Nottingham · PhD
Considered and rejected
Considered and rejected: Excluded recombinant proteins EscF, EspF, and AnsB from the initial in vitro DNA digestion screening because they were either not expressed or formed insoluble inclusion bodies in E. coli BL21(DE3).
THE ROLE OF ATTACHING AND EFFACING PATHOGENS IN PROMOTING LETHAL DIARRHEA AND LONG-TERM COLON TUMORIGENESIS · Johns Hopkins · PhD
Tried and failed
recombinant protein expression and truncation constructs applied to large membrane-associated bacterial adhesin protease. Outcome: unstable. Reason: both full-length and truncated constructs suffered irreversible aggregation upon purification
COMPARATIVE STRUCTURE-FUNCTION ANALYSIS OF ORTHOLOGOUS SYSTEMS OF LARGE ADHESIN REGULATION IN BACTERIAL BIOFILM FORMATION · Cornell · PhD
Tried and failed
recombinant bacterial protein expression applied to full-length viral coat protein. Reason: expressed protein formed insoluble aggregates upon cell lysis
THE MASTER MANIPULATOR; CRYSTAL CLEAR PERSPECTIVES ON VIRAL INFECTIVITY · Cornell · PhD
Tried and failed
recombinant expression in bacterial and insect systems applied to full-length eukaryotic multidomain protein. Reason: expressed protein formed insoluble aggregates and underwent extensive proteolysis
Structural and biochemical studies of RAD51 filament modulators · Imperial · PhD
Tried and failed
recombinant bacterial expression with fusion tags and truncations applied to membrane-associated protein. Reason: protein remained completely insoluble across multiple expression vectors, buffer conditions, and N-terminal transmembrane truncations
A small molecule-guided structure-function exploration of mitochondrial aldehyde dehydrogenase · MIT · PhD
Target constructs fail to express or suffer severely diminished yields
Heterologous expression constructs repeatedly yielded no detectable protein or failed across mammalian, insect, and bacterial expression systems. Specific mutations, altered domain boundaries, or multi-protein complex requirements led to negligible production and prevented target isolation.
Tried and failed
recombinant protein expression and purification applied to bacterial effector protein. Reason: protein failed to express in soluble form under standard conditions
Antimicrobial Dynamics of the Vibrio cholerae Type VI Secretion System · Georgia Tech · PhD
Tried and failed
recombinant protein complex expression and affinity purification applied to heterodimeric protein complex in mammalian cells. Reason: complex could not be expressed and purified despite multiple tagged constructs
AMPK and FLCN/FNIP: investigating regulation, interactions and signalling crosstalk · Imperial · PhD
Tried and failed
recombinant operon co-expression and affinity purification applied to bacterial defense multi-protein complexes. Reason: proteins failed to express or purify from heterologous bacterial host
Supramolecular complex formation in bacterial anti-phage defense and viral immune evasion · Harvard · PhD
Tried and failed
recombinant protein expression and purification applied to full-length lipid-binding protein and isolated domains. Reason: unable to successfully purify the isolated C-terminal domains or full-length protein for in vitro analysis
FROM MECHANISM TO THERAPEUTICS: DECIPHERING THE ROLES OF A NOVEL LIPID-BINDING PROTEIN IN WNT SIGNALING AND CANCER · Cornell · PhD
Tried and failed
recombinant protein expression in bacterial host applied to heterologous eukaryotic transcription factors. Reason: complete lack of protein expression across multiple construct designs and growth conditions
Investigation of Protein Conformation With Novel Labeling Techniques Using Electron Paramagnetic Resonance Spectroscopy · Cornell · PhD
Tried and failed
baculovirus-mediated recombinant protein expression applied to heterodimeric protein complexes. Reason: expression of the protein complexes in insect cells was unsuccessful
Considered and rejected
Considered and rejected: Rejected expressing OaAEP1 completely without its C-cap because recombinant expression was unsuccessful.
Investigation of Protein Conformation With Novel Labeling Techniques Using Electron Paramagnetic Resonance Spectroscopy · Cornell · PhD
Considered and rejected
Considered and rejected: Rejected combining multiple engineered disulfide bonds or pairing S884C/A893C with proline substitution F817P due to reduced recombinant expression yields
Targeting viral fusion for immunity and therapeutics against infection · UT Austin · PhD
Tried and failed
framework backmutation during antibody humanisation applied to humanised Fab fragment expression. Reason: a specific framework backmutation abrogated recombinant protein expression completely
The humanisation and generation of antibody fragments against oxidised low density lipoprotein for the optical molecular imaging of atherosclerosis · Imperial · PhD
Expressed proteins suffer from misfolding, defective post-translational processing, or improper assembly
Recombinant proteins expressed in bacterial hosts often lacked critical modifications such as glycosylation and disulfide bonds, leading to misfolding and non-specific binding. Additionally, fusion proteins and engineered constructs displayed polydispersity, failed membrane trafficking, or formed inconsistent assemblies that did not match native benchmarks.
Tried and failed
recombinant fusion protein crystal encapsulation applied to heterologous spore-forming bacterial expression systems. Outcome: did not generalise. Reason: fusion proteins failed to form extracellular crystals when expressed in non-native bacterial host strains
Spore-forming bacteria as pharmaceutical factories · Cambridge · PhD
Lost to a baseline
Recombinant globular protein rBLG50 showed lower interfacial coverage and stability compared to native bovine Whey Protein Isolate and α-casein benchmarks.
Two-phase microfluidic approaches to study food protein functionality · Texas Tech · PhD
Tried and failed
recombinant protein expression with fusion tag applied to amyloid fibril preparation. Reason: yielded inconsistent fibril assembly resulting in mismatched solid-state NMR spectra
Structural Characterization of Plaque Seeded Amyloid-β Fibrils by Magic Angle Spinning NMR · MIT · PhD
Tried and failed
Bacterial expression of complex mammalian lectins applied to recombinant protein production. Reason: Lack of N-glycosylation and disulfide bond formation caused misfolding and non-specific binding.
Carbohydrate and bacterial binding specificity of human intelectin-1 · MIT · PhD
Tried and failed
recombinant purification of full-length coiled-coil protein applied to bacterial cell wall regulatory proteins. Outcome: unstable. Reason: the coiled-coil region caused polydispersity and aggregation in the apo state
Unifying regulatory mechanism of bacterial cell wall synthesis · Harvard · PhD
Tried and failed
heme precursor supplementation in culture media applied to membrane protein expression and cell viability. Reason: failed to improve host cell health or recombinant protein membrane trafficking
The Development Of Novel Genetically Encoded Voltage Indicators Using De Novo Designed Proteins · Penn · PhD
Downstream purification is impaired by persistent co-purification of host contaminants
Target proteins co-purified persistently with endogenous host membrane transporters, chaperone proteins, and bacterial nucleic acids despite high-stringency washes. Standard chromatography workflows lacked sufficient resolution to eliminate impurities or persistent bacterial endotoxin contamination above acceptable limits.
Tried and failed
inclusion body prep with ion exchange chromatography applied to multiply modified recombinant proteins. Reason: standard chromatography steps lacked sufficient resolving power to yield pure modified target protein
Investigation of parameters of chromatin higher order structure and the making of multiply-acetylated histone H3 via nonsense suppression · Iowa State · PhD
Considered and rejected
Considered and rejected: Rejected using bacterial recombinant expression for in vivo affibody applications due to persistent endotoxin contamination above FDA device limits, switching to solid-phase peptide synthesis (SPPS).
Engineering Shelf-Stable Affibody-Functionalized Hydrogels for Controlled Dual Release of Bone Morphogenetic Protein-2 and Interleukin-4 · University of Oregon · PhD
Considered and rejected
Considered and rejected: Purification of recombinant RTP4 N-terminal ZFD directly from bacteria without denaturing/refolding failed because it co-purified with bacterial nucleic acids and formed insoluble aggregates.
Identification of Receptor Transporting Proteins As Conserved Antiviral Effectors in Vertebrates · UT Southwestern · PhD
Tried and failed
recombinant protein expression in wild-type host applied to membrane protein purification. Reason: endogenous host membrane transporter co-purified persistently despite high-stringency affinity washes
Investigation of the oligomeric state of the mycobacterial lipid transporter, MmpL3: biophysical and computational insights · Imperial · PhD
Heterologous expression induces host cell toxicity and suppresses organism growth
Expression of heterologous proteins caused severe toxicity to host cells, resulting in transformation failure or in vitro refolding failure. Recombinant strains also exhibited significantly diminished biomass accumulation and impaired cell growth compared to parental counterparts.
Tried and failed
recombinant bacterial protein expression and inclusion refolding applied to large parasite structural protein domain. Reason: host cell toxicity upon induction, inclusion body formation, and failed in vitro refolding despite solubility tags
Structural biology of T. gondii glideosome-associated Connector (TgGAC) · Imperial · PhD
Lost to a baseline
Biomass yield of recombinant strain rSZ No. 8 at 120 h (27.81 ± 1.75 g/L) was significantly lower than the parent strain B3 (31.36 ± 1.57 g/L, P < 0.05).
Schizochytrium microalgae expressing recombinant bovine lactoferrin as a route to improve intestinal health in calves · University College Dublin · PhD
Tried and failed
co-expressing recombinant single-stranded DNA binding proteins applied to heterologous bacterial recombineering. Reason: heterologous single-stranded DNA binding protein expression caused severe host toxicity and transformation failure
Developing and characterizing phage-encoded homologous recombination machinery as a genome editing tool · Harvard · PhD
Left open by the authors
Problems the authors named and did not get to.
Left open
Perform structural and functional analyses to verify the integrity of recombinant HAP40 purified from E. coli inclusion bodies. Blocker: Requires a wet biology lab and physical protein samples purified from bacterial inclusion bodies
Left open
Recombinantly generate and express the identified surface-accessible EV protein candidates in a biological expression system. Blocker: Requires a wet laboratory for molecular cloning, recombinant protein expression, and cell culture
Identification of surface accessible proteins in parasitic nematode extracellular vesicles · Iowa State · Master's
Left open
Recombinantly express and functionally validate plasma monoclonal antibodies from lineages exclusively detected in IC-Seq to identify effector functions. Blocker: Requires wet-lab facilities for recombinant protein expression and functional antibody effector assays
Elucidating properties of the plasma antibody repertoire response following erythrocytic-stage malaria vaccination and infection · UT Austin · PhD
Left open
Perform cell-free in vitro reconstitution of R-body synthesis using combinatorial expression of Reb A, B, C, and D. Blocker: Requires a wet lab, recombinant proteins/plasmids, and cell-free protein expression systems.
An Interdisciplinary Investigation of Conformational Changes in Quasi-Crystalline Protein Array R-bodies in Response to pH · University of Washington · PhD
Left open
Express and incorporate full-length soluble IgG1 Fab domain split-intein fusion proteins into multi-part protein trans-splicing assembly. Blocker: Requires wet lab equipment, recombinant protein expression, and biochemical trans-splicing assays
Towards the decentralized manufacturing of multi-domain proteins · Imperial · PhD
Left open
Encapsulate ELP-fused deoxyviolacein pathway enzymes (VioC and VioE) or membrane proteins into protein vesicles to construct artificial organelles. Blocker: Requires wet lab biochemical synthesis, recombinant fusion protein expression, and membrane protein reconstitution
Engineering fusion protein vesicles for biocatalysis and drug delivery applications · Georgia Tech · PhD
Left open
Heterologously express and assemble a functional recombinant ammonia monooxygenase (AMO) holoenzyme complex in E. coli. Blocker: Requires a wet biology lab, specialized expression vectors, bacterial culturing, and biochemical purification equipment
AN INVESTIGATION INTO COPPER PROTEINS GERMANE TO BIOLOGICAL AMMONIA OXIDATION · Cornell · PhD
Left open
Produce functional NA tetrameric head coating antigens to enhance ELISA detection breadth and preserve conformational epitopes. Blocker: Requires wet lab recombinant protein expression, purification, and biochemical validation apparatus
Investigation on the Immunogenicity of Neuraminidase and Optimization of Antigen Display for Influenza Vaccines · Harvard · Master's
Left open
Characterise and compare recombinant in vitro FapC homo-amyloids with natively secreted hetero-amyloids. Blocker: Requires wet-lab biochemistry, protein expression, and structural characterization facilities.
Structural and functional insights into Functional Amyloid biogenesis in Pseudomonas · Imperial · PhD
Who has worked on this
Authors of the public theses cited on this page, most-cited first.
- Robert Dunleavy · Cornell · PhD · 2023
Investigation of Protein Conformation With Novel Labeling Techniques Using Electron Paramagnetic Resonance Spectroscopy - Jennifer Chigozie Okoye · Nottingham · PhD · 2021
The interaction of glycosylated major outer membrane protein of Campylobacter jejuni with natural and synthetic ligands - Varun Potdar · Texas Tech · PhD · 2026
Two-phase microfluidic approaches to study food protein functionality - Yingkun Zhu · University College Dublin · PhD · 2026
Schizochytrium microalgae expressing recombinant bovine lactoferrin as a route to improve intestinal health in calves - Xu Zhang · Imperial · PhD · 2025
Structural biology of T. gondii glideosome-associated Connector (TgGAC) - Sadie Priscilla Antine · Harvard · PhD · 2025
Supramolecular complex formation in bacterial anti-phage defense and viral immune evasion - Alexander Laughlin · Cornell · PhD · 2025
AN INVESTIGATION INTO COPPER PROTEINS GERMANE TO BIOLOGICAL AMMONIA OXIDATION - Irina Shlosman · Harvard · PhD · 2025
Unifying regulatory mechanism of bacterial cell wall synthesis - Jonathan Dorogin · University of Oregon · PhD · 2025
Engineering Shelf-Stable Affibody-Functionalized Hydrogels for Controlled Dual Release of Bone Morphogenetic Protein-2 and Interleukin-4 - Sara Cioccolo · Imperial · PhD · 2024
Investigation of the oligomeric state of the mycobacterial lipid transporter, MmpL3: biophysical and computational insights - Johanna Sophie Rehfeld · Leibniz Universität Hannover · PhD · 2024
Validierung von 3D-gedruckten Reaktorsystemen für biotechnologische Applikationen - Enzo Morro · EPFL · PhD · 2024
Exploring the therapeutic potential of Huntingtin native state stabilization through targeting its interacting partners
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