Biology subjects

Singh, J.

Publications and source records attributed to Singh, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Spatial profiling identifies regionally distinct microenvironments and targetable immunosuppressive mechanisms in pediatric osteosarcoma pulmonary metastases.

Osteosarcoma is the most common malignant bone tumor in young patients and remains a significant clinical challenge, particularly in the context of metastatic disease. Despite extensive documentation of genomic alterations in osteosarcoma, studies detailing the immunosuppressive mechanisms within the metastatic osteosarcoma microenvironment are lacking. Our objective was to characterize the spatial transcriptional landscape of metastatic osteosarcoma to reveal these immunosuppressive mechanisms and identify promising therapeutic targets. Here, we performed spatial transcriptional profiling on a cohort of osteosarcoma pulmonary metastases from pediatric patients. We reveal a conserved spatial gene expression pattern resembling a foreign body granuloma, characterized by peripheral inflammatory signaling, fibrocollagenous encapsulation, lymphocyte exclusion, and peritumoral macrophage accumulation. We also show that the intratumoral microenvironment of these lesions lack inflammatory signaling. Additionally, we identified CXCR4 as an actionable immunomodulatory target that bridges both the intratumoral and extratumoral microenvironments and highlights the spatial heterogeneity and complexity of this pathway. Collectively, this study reveals that metastatic osteosarcoma specimens are comprised of multiple regionally distinct immunosuppressive microenvironments.

cancer biology↗

bioRxiv2025

Integrative spatial transcriptomic analysis pinpoints the role of TaMCO3 encoding ferroxidase in wheat root tip iron mobilization

Roots play an critical role in the sensing and absorption of essential minerals from the rhizosphere. Iron (Fe) deficiency, for example, triggers a well-known series of physiological and molecular responses within roots that facilitate uptake, which differs between monocots and dicots. In monocots, little is known about molecular responses that occur within specific root development zones in response to iron deprivation, and how these differences results in overall nutrient uptake. Here, we conducted a transcriptome analysis of wheat root tips under Fe deficiency (-Fe) and performed a comparative transcriptome analysis with the previous datasets generated from the whole root. Gene ontology analysis of differentially expressed genes highlighted the significance of oxidoreductase activity and metal/ion transport in the root tip, which are critical for Fe mobilisation. Interestingly, wheat, an allohexaploid species consisting of three different genomes (A, B, and D) displayed varying gene expression levels arising from the three genomes that contributed to similar molecular functions. Detailed analysis of oxidoreductase function at the root tip revealed multiple multi-copper oxidase (MCO) proteins, such as Fe-responsive TaMCO3, that likely contribute to the overall ferroxidase activity. Detailed characterisation of TaMCO3 shows that it complements the yeast FET3 mutant and rescues the -Fe sensitivity phenotype of Arabidopsis atmco3 mutants by enhancing vascular Fe loading. Transgenic wheat lines overexpressing TaMCO3 exhibited increased root Fe accumulation and improved tolerance to -Fe by augmenting the expression of Fe-mobilizing genes. Our findings highlight the role of spatially resolved gene expression in -Fe responses, suggesting strategies to reprogram cells for improved nutrient stress tolerance.

plant biology↗

bioRxiv2025

Inferring single-cell dynamics of a molecular reporter from unlabeled live-cell light microscopy data analyzed with delay embedding

Quantification of the temporal sequence of molecular behavior in live individual cells holds promise for improving causal and mechanistic models of cell biology. In recent years, different methods for inferring molecular labeling from microscopy data have been developed, especially in the context of "virtual pathology", but less effort has been directed to the context of single-cell dynamics and live-cell imaging. We demonstrate that phase-contrast live-cell imaging of MCF10A cells, without labeling data, is predictive of dynamical, single-cell behavior of the cell-cycle reporter Human DNA Helicase B (HDHB) - in particular, of the nuclear vs. cytoplasmic localization of this fluorescent reporter of cyclin-dependent kinase activity. Prediction quality improves substantially when temporal sequences of images are combined in a "delay embedding" framework. When different featurizations of the imaging data are examined, we find that features derived from a variational auto-encoder (VAE) outperform "classical" image features derived from shape and texture. We find the best performance, with Pearson R[~] 0.9 on test data, using VAE features augmented by categorical predictions, all within the delay-embedding framework - in comparison to R [~] 0.5 based on "ordinary" regression with VAE features.

cell biology↗

bioRxiv2025

An intranasal, NLC-delivered self-amplifying RNA vaccine establishes protective immunity against pre-pandemic H5N1 and H7N9 influenza

Seasonal and pandemic influenzas are continuous threats to human health, requiring rapid development of vaccines to multiple evolving viral strains. New RNA vaccine technologies have the adaptability and manufacturability to facilitate pandemic preparedness but have limited flexibility in their route of administration, reducing the ability to establish local protective immune responses such as respiratory mucosal immunity. Here, we describe monovalent and bivalent self-amplifying RNA (saRNA) vaccines against A/Vietnam/1203/2004 H5N1 and A/Anhui/2013 H7N9. These saRNA vaccines express either H5 or H7 hemagglutinin and are formulated with a nanostructured lipid carrier (NLC) that permits both intramuscular (IM) and intranasal (IN) dosing. In mice, IM vaccination established systemic humoral and cellular responses but no detectable mucosal response, while IN administration induced robust systemic and mucosal immunity. The saRNA-NLC vaccines provided complete protection against morbidity and mortality in ferret challenge models, establishing this intranasally-administered saRNA-NLC vaccine platform as a potential pandemic response tool. ONE SENTENCE SUMMARYA self-amplifying RNA-NLC vaccine, delivered intranasally, induces robust mucosal immunity in mice and protects against H5N1 and H7N9 in ferrets

immunology↗

bioRxiv2024

Notch and LIM-homeodomain protein Arrowhead regulate each other in a feedback mechanism to play a role in wing and neuronal development in Drosophila

Notch pathway is an evolutionarily conserved signaling system that operates to influence an astonishing array of cell fate decisions in different developmental contexts. To identify novel effectors of Notch signaling, we analyzed the whole transcriptome of Drosophila wing and eye imaginal discs in which an activated form of Notch was overexpressed. A LIM homeodomain protein Arrowhead (Awh) was identified as a novel candidate which plays a crucial role in Notch mediated developmental events. Awh alleles show strong genetic interaction with Notch pathway components. Awh loss-of-function upregulates Notch targets Cut and Wingless. Awh gain-of-function downregulates Notch targets by reducing the expression of ligand, Delta. Consequently, the expression of Wingless effector molecule Armadillo and its downstream targets, Senseless and Vestigial, also gets downregulated. Awh overexpression leads to ectopicexpression of engrailed, a segment polarity gene in the anterior region of wing disc, leading to patterning defects. Additionally, Notch gain-of-function mediated neuronal defects get significantly rescued with Awh overexpression. Activated Notch inhibits Awh activity, suggesting a regulatory loop between Awh and Notch. Additionally, the defects caused by Awh gain-of-function were remarkably rescued by Chip, a LIM interaction domain containing transcriptional co-factor. The present study highlights the novel feedback regulation between Awh and Notch.

developmental biology↗

bioRxiv2024

A conserved cell-type gradient across the human mediodorsal and paraventricular thalamus

The mediodorsal thalamus (MD) and adjacent midline nuclei are important for cognition and mental illness, but their cellular composition is not well defined. Using single-nucleus and spatial transcriptomics, we identified a conserved excitatory neuron gradient, with distinct spatial mapping of individual clusters. One end of the gradient was expanded in human MD compared to mice, which may be related to the expansion of granular prefrontal cortex in hominids. Moreover, neurons preferentially mapping onto the parvocellular division MD were associated with genetic risk for schizophrenia and bipolar disorder. Midbrain-derived inhibitory interneurons were enriched in human MD and implicated in genetic risk for major depressive disorder.

neuroscience↗

bioRxiv2024

Behavioral screening defines three molecular Parkinsonism subgroups in Drosophila

Parkinsonism is defined by motor dysfunction, but the specific upstream molecular causes of these clinical symptoms can vary widely. We hypothesize that these causes converge onto a limited number of core cellular pathways. To investigate this, we created a new collection of 24 genetically very well-controlled animal models of familial forms of parkinsonism. Using unbiased behavioral screening and machine learning we identified three clusters of mutants that converge on (1) mitochondrial function; (2) retromer/vesicle trafficking; and (3) proteostasis/autophagy. Genes within each cluster have a similar genetic interaction profile and compounds that target specific molecular pathways ameliorate dopaminergic neuron dysfunction in a cluster-specific manner. This suggests that familial parkinsonism can be stratified into three broad functional groups and our findings pave the way for targeted biomarker discovery and drug development.

neuroscience↗

bioRxiv2024

Contribution of brain intrinsic branched-chain amino acid metabolismin a novel mouse model of maple syrup urine disease

Maple syrup urine disease (MSUD) results from loss of branched-chain ketoacid dehydrogenase (BCKDH) activity, the committed, rate-limiting step of branched-chain amino acid (BCAA) oxidation. Current treatments, including a low protein diet and liver transplantation, improve peripheral biochemistry and limit episodes of metabolic decompensation but do not fully prevent chronic neuropsychiatric symptoms. The mechanisms underlying chronic neurologic phenotypes remain poorly understood. Currently available MSUD mouse models do not survive long enough to evaluate chronic central nervous system (CNS) pathology. To investigate if loss of brain-intrinsic BCAA metabolism contributes to chronic neurologic disease, we developed a new brain-specific knockout mouse model of MSUD. First, we generated a mouse harboring a floxed Dbt allele (Dbtflox/flox). Then we crossed this line with Cre recombinase driver lines to induce loss of Dbt expression in 1) all developing CNS cell populations 2) neurons alone or 3) astrocytes alone. We found that brain-specific KO mice have elevations in BCAA levels in cortex that are exacerbated by a high protein diet. They also have secondary changes in amino acids that are important for neuronal function, including glutamine and glycine. These metabolic differences result in subtle functional deficits as measured by electroencephalogram and behavioral testing. Astrocyte and neuron-specific KO mice each also demonstrate mild biochemical features of MSUD in the cortex, suggesting that both cell populations may contribute to disease pathology. Collectively, these data suggest that therapies targeting the CNS directly, in addition to the periphery, may improve outcomes in MSUD.

genetics↗

bioRxiv2024

Discordance in Genotypic and Phenotypic anti-tuberculosis drug susceptibility results: time to reconsider critical concentration.

ObjectiveTo correlate rpoB mutations found on the sanger sequencing in Mycobacterium tuberculosis (MTB) isolates with Minimum Inhibitory Concentrations (MICs) to the rifampicin. MethodsWe assessed the minimum inhibitory concentrations (MICs) for 151 archived clinical MTB isolates that were determined phenotypically susceptible to RIF (101;66.89%) and remaining fifty (50;33.11%) were resistant to RIF by BACTEC MGIT SIRE DST. MIC values were determined using colorimetric redox indicator (Resazurin/REMA) method and results were correlated with rpoB gene mutations associate with rifampicin resistance found. ResultsComparing the MIC and critical concentration, we found that 15 of these 101 (14.85%) isolates were misclassified by MGIT-960 as sensitive at standard critical concentration (1.0{micro}g/mL) though these were found to have low-level RIF resistance by CRI assay (MIC 0.50{micro}g/mL to 1.0{micro}g/mL) and sanger sequencing. We found that all of 15 isolates contained non-synonymous mutations, the commonest being the Ile572Phe (7, 46.66%), followed by Leu533Pro (3, 20.0%), His526Leu (2, 13.33%), His526Asn+Ile572Phe (1), Asp516Tyr (1), and Leu533Pro+Pro564Arg (1). These mutations are reported to confer low-level RIF resistance. But we did not find any mutation at MIC <0.25g/mL. ConclusionWe found that a significant number of MTB isolates have phenotypic and genotypic discordance. Taking 1.0{micro}g/mL of rifampicin as critical concentration, isolates from approximately 15% patients are misidentified as susceptible to rifampicin, even when these strains carry low level drug resistance conferring mutations and have potential to develop clinical MDR-TB.

microbiology↗

bioRxiv2024

Dispensable genome and segmental duplications drive the genome plasticity in Fusarium solani

Fusarium solani is a species complex encompassing a large phylogenetic clade with diverse members occupying varied habitats. We recently reported a unique opportunistic F. solani associated with unusual dark galls in sugarbeet. We assembled the chromosome-level genome of the F. solani sugarbeet isolate strain SB1 using Oxford Nanopore and Hi-C sequencing. SB1 has a large genome (59.38 Mb) organized into 15 chromosomes. The genome expansion is due to the high repeats and massive segmental duplications within its three potentially accessory chromosomes. These chromosomes are absent in the closest reference genome with chromosome-level assembly, F. vanettenii 77-13-4. The extensive segmental duplications between the two SB1 chromosomes suggest that this isolate may have doubled its accessory genes. Further comparison of the F. solani strain SB1 genome demonstrates inversions and syntenic regions to an accessory chromosome of F. vanettenii 77-13-4. The pan-genome of 12 publicly available F. solani isolates nearly reached gene saturation, with few new genes discovered after the addition of the last genome. Based on orthogroups and average nucleotide identity, F. solani is not grouped by lifestyle or origin. The pan-genome analysis further revealed the enrichment of several enzymes-coding genes within the dispensable (accessory + unique genes) genome, such as hydrolases, transferases, oxidoreductases, lyases, ligases, isomerase, and dehydrogenase. The evidence presented here suggests that genome plasticity, genetic diversity, and adaptive traits in Fusarium solani are driven by the dispensable genome with significant contributions from segmental duplications.

genomics↗

bioRxiv2024

Generating human AMN and cALD iPSC-derived astrocytes with potential for modeling X-linked adrenoleukodystrophy phenotypes

X-adrenoleukodystrophy (X-ALD) is a peroxisomal metabolic disorder caused by mutations in the ABCD1 gene encoding the peroxisomal ABC transporter adrenoleukodystrophy protein (ALDP). Similar mutations in ABCD1 may result in a spectrum of phenotypes in males with slow progressing adrenomyeloneuropathy (AMN) and fatal cerebral adrenoleukodystrophy (cALD) dominating the majority of cases. Mouse model of X-ALD does not capture the phenotype differences and an appropriate model to investigate mechanism of disease onset and progress remains a critical need. Induced pluripotent stem cell (iPSC)-derived and cell models derived from them have provided useful tools for investigating cell-type specific disease mechanisms. Here, we generated induced pluripotent stem cell (iPSC) lines from skin fibroblasts of two each of apparently healthy control, AMN and cALD patients with non-integrating mRNA-based reprogramming. iPSC lines expanded normally and expressed pluripotency markers Oct4, SOX2, Nanog, SSEA and TRA-1-60. Expression of markers SOX17, brachyury, Desmin, Oxt2 and beta tubulin III demonstrated the ability of the iPSCs to differentiate into all three germ layers. iPSC-derived lines from CTL, AMN and cALD male patients were differentiated into astrocytes. Differentiated AMN and cALD astrocytes lacked ABCD1 expression and accumulated VLCFA, a hallmark of X-ALD. These patient astrocytes provide disease-relevant tools to investigate mechanism of differential neuroinflammatory response and metabolic reprogramming in X-ALD. Further these patient-derived human astrocyte cell models will be valuable for testing new therapeutics.

neuroscience↗

bioRxiv2024

Scribble knockdown induced metastasis tracking, identification of its associated novel molecular candidates through Proteome and in silico studies

Metastasis is the primary cause of cancer associated death globally. Loss of function of Scribble, a cell polarity regulator/tumor suppressor gene, is associated with many forms of human cancer but its role in cell proliferation and metastasis remains unknown. We generated metastatic cancer in Drosophila using UAS-GAL4 system, through knockdown of Scribble in the wing imaginal discs and tracked metastasis events from 0hr early pupae to 84hrs late pupae using fluorescence microscope. Here, we report, for the first time, that knockdown of Scribble alone could lead to the development of primary tumor in the wing imaginal discs, which is capable of establishing metastasis, leading to secondary tumor formation, eventually resulting in absolute pupal lethality. Further, we checked a metastasis biomarker, MMP1 levels during pre-and post-metastatic phases in Drosophila pupae using qRT-PCR and Western blot analysis. In addition, we analyzed the proteome of Scribble knockdown induced tumor-bearing pupae by 2-D gel electrophoresis followed by MALDI-TOF MS to identify novel proteins involved in the process of tumorigenesis and metastasis. We identified six differentially expressed proteins, Obp 99b, Fer2LCH,CG13492, Hsp23, Ubiquitin and Colt in Scrib knockdown pupae compared to wild-type and validated their expression at the transcriptional level using qRT-PCR. In-silico studies show these novel protein interaction with Scrib. Thus, our results suggested that loss of Scrib alone causes metastasis, without the need for cooperative interaction with oncogenic Ras. The newly identified Fer2LCH (ferritin) and colt proteins could be important candidates for therapeutic target against Scrib associated cell proliferation and metastasis.

cancer biology↗

bioRxiv2024

Drp1-JNK knockdown mitigates Scribble loss induced cell proliferation, metastasis and lethality phenotypes in Drosophila

Mitochondrial dynamics are emerging as master regulators for targeting several types of cancers, including breast cancer, cervical cancer, and hepatocellular carcinoma, for therapeutic intervention. Mitochondrial morphology, size, position and activity within cells is regulated by dynamic fission and fusion events. Dynamin-related protein 1 (Drp1) promotes mitochondrial fission and maintains mitochondrial homeostasis. Loss of Scrib is implicated in several human cancers wherein mitochondrial dysfunction leads to excessive cell proliferation and metastasis. However, the exact molecular mechanisms behind the Scrib loss induced dysregulation of mitochondrial dynamics in cancer progression remains obscure. Although the role of mitochondrial dynamics are being investigated in several types of cancers, but the role of Drp1- mediated fission event in regulating the maintenance of polarity of cells upon loss of Scrib function is elusive. In this study, for the first time, we blocked the function of Drp1 activity in Scrib knockdown induced metastasis cancer model by two ways, firstly, through genetic ablation of Drp1, and secondly by using mdivi-1, a Drp1 specific inhibitor. Genetic depletion of Drp1 expression (Drp1RNAi) in Scrib knockdown cells inhibits Metalloproteinase MMP1, reduces ROS production, restores apico-basal (A/B) cell polarity and enhances ATP production. Further to confirm role of Drp1 in regulation of cell polarity, we employed mdivi, a Drp1 specific inhibitor which has dose dependent effect in cell polarity regulation. This study also reveals that JNK inhibition (JNKRNAi) in Scrib abrogated cells mitigates the Drp1 expression and controls cell proliferation leading to restoration of mitochondrial morphology and epithelial cellpolarity. Our results highlight Drp1 as a key regulator in maintaining the apico-basal polarity of cells which gets affected upon loss of Scrib but Drp1-JNK downregulation effectively mitigates ScribRNAi associated cell proliferation, metastasis and pupal lethality phenotypes.

cancer biology↗

bioRxiv2024

PPARγ mediated enhanced lipid biogenesis fuels Mycobacterium tuberculosis growth in hepatocytes.

Mycobacterium tuberculosis (Mtb) infection of the lungs, besides producing prolonged cough with mucus, also causes progressive fatigue and cachexia with debilitating loss of muscle mass. While anti-tuberculosis (TB) drug therapy is directed toward eliminating bacilli, the treatment regimen ignores the systemic pathogenic derailments that probably dictate TB-associated mortality and morbidity. Presently, it is not understood whether Mtb spreads to metabolic organs and brings about these impairments. Here we show that Mtb creates a replication-conducive milieu of lipid droplets in hepatocytes by upregulating transcription factor PPAR{gamma} and scavenging lipids from the host cells. In hepatocytes, Mtb shields itself against the common anti-TB drugs by inducing drug-metabolizing enzymes. Infection of the hepatocytes in the in vivo aerosol mice model can be consistently observed post-week 4 along with enhanced expression of PPAR{gamma} and drug-metabolizing enzymes. Moreover, histopathological analysis indeed shows the presence of Mtb in hepatocytes along with granuloma-like structures in human biopsied liver sections. Hepatotropism of Mtb during the chronic infectious cycle results in immuno-metabolic dysregulation that could magnify local and systemic pathogenicity, altering clinical presentations.

microbiology↗

bioRxiv2024

Translation initiation or elongation inhibition triggers contrasting effects on Caenorhabditis elegans survival during pathogen infection

Diverse microbial pathogens are known to attenuate host protein synthesis. Consequently, the host mounts a defense response against protein translation inhibition, leading to increased transcript levels of immune genes. The seemingly paradoxical upregulation of immune gene transcripts in response to blocked protein synthesis suggests that the defense mechanism against translation inhibition may not universally benefit host survival. However, a comprehensive assessment of host survival on pathogens upon blockage of different stages of protein synthesis is currently lacking. Here, we investigate the impact of knockdown of various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, we observe opposing effects on C. elegans survival depending on whether translation initiation or elongation is inhibited. While translation initiation inhibition enhances survival, elongation inhibition decreases it. Transcriptomic studies reveal that translation initiation inhibition activates a bZIP transcription factor ZIP-2-dependent innate immune response that protects C. elegans from P. aeruginosa infection. In contrast, inhibiting translation elongation triggers both ZIP-2-dependent and ZIP-2-independent immune responses that, while effective in clearing the infection, are detrimental to the host. Thus, our findings reveal the opposing roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection, highlighting distinct transcriptional reprogramming that may underlie these differences.

immunology↗

bioRxiv2024

Genomes of Aegilops umbellulata provide new insights into unique structural variations and genetic diversity in the U-genome for wheat improvement

Aegilops spp. serve as an important reservoir for novel sources of resistance or tolerance to biotic and abiotic stresses. To harness this reservoir, we have generated a high-quality chromosome-level genome assembly of an Ae. umbellulata accession PI 554389 using a combination of PacBio HiFi, Oxford nanopore, and chromosome conformation capture (Hi-C) sequencing technologies and resequenced 20 Ae. umbellulata genomes using Illumina sequencing. We assembled a 4.20 Gb genome spanned over seven chromosomes, rich in repetitive elements ([~]84%), achieving a QV of 59.54 with 98.14% completeness. The phylogenetic analysis places the U-genome with D-lineage, but major and distinct rearrangements were revealed in the U-genome. Unique transposon landscape of diploid U-genome and complex chromosomal rearrangements, most prominently in 4U and 6U chromosomes uncovered a distinct evolutionary trajectory of Ae. umbellulata. Additionally, the resequencing of geographically and morphologically diverse Ae. umbellulata accessions revealed three distinctive evolutionary sub-populations. Resequencing also identified six new haplotypes for Lr9, the first leaf rust resistance gene introgressed and cloned from Ae. umbellulata. These genomics resources along with high levels of resistance in the resequenced accessions against five devastating wheat diseases affirmed the genetic potential of Ae. umbellulata for wheat improvement.

plant biology↗

bioRxiv2023

Inhibition of the UFD-1-NPL-4 complex triggers an inflammation-like response in Caenorhabditis elegans

The UFD-1 (ubiquitin fusion degradation 1)-NPL-4 (nuclear protein localization homolog 4) heterodimer is involved in extracting ubiquitinated proteins from several plasma membrane locations, including the endoplasmic reticulum. This heterodimer complex helps in the degradation of ubiquitinated proteins via the proteasome with the help of the AAA+ ATPase CDC-48. While the ubiquitin-proteasome system is known to have important roles in maintaining innate immune responses, the role of the UFD-1-NPL-4 complex in regulating immunity remains elusive. In this study, we investigate the role of the UFD-1-NPL-4 complex in maintaining Caenorhabditis elegans innate immune responses. Inhibition of the UFD-1-NPL-4 complex activates an aberrant immune response that reduces the survival of the wild-type worms on the pathogenic bacterium Pseudomonas aeruginosa despite diminishing colonization of the gut with the bacterium. This aberrant immune response improves the survival of severely immunocompromised worms on pathogenic bacteria but is detrimental on nonpathogenic bacteria. Transcriptomics studies reveal that the GATA transcription factor ELT-2 mediates the aberrant immune response upon inhibition of the UFD-1-NPL-4 complex. Collectively, our findings show that inhibition of the UFD-1-NPL-4 complex triggers an aberrant immune response that is detrimental to immunocompetent worms under infection conditions but can be advantageous for immunocompromised worms.

immunology↗

bioRxiv2023

The Expression of HIV-1 Tat in Lactococcus lactis

Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis.

microbiology↗