RESUMEN

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) is a rare and highly aggressive lymphoma with characteristic ALK rearrangements. Various fusion genes involving ALK have been demonstrated, but the influence of the ALK fusion partners on ALK protein expression and the genetic characteristics of ALK+ LBCL remain relatively unknown. In this study, we conducted an extensive clinicopathological and molecular analysis on seven cases of ALK+ LBCL to explore the correlation between ALK fusion genes and ALK protein expression, thereby enriching the genetic characteristics of this tumour. We integrated the findings from clinical, histopathological/immunophenotypic, and molecular studies, including three samples subjected to next-generation sequencing, and six cases underwent RNA-based ALK fusion gene detection. We identified five distinct types of ALK fusion genes, including CLTC, NPM1, PABPC1, SEC31A, and TFG. Notably, only the NPM1::ALK fusion showed nuclear and cytoplasmic ALK staining, and the remaining four fusion genes resulted in cytoplasmic ALK staining. Our analysis revealed that the CLTC::ALK fusion resulted in a unique cytoplasmic perinuclear Golgi zone focal granular heterogeneous staining pattern of ALK. Additionally, we identified six potentially clinically significant gene mutations, including TET2, CHD2, DTX1, KMT2D, LRP1B, and XPO1. Furthermore, in all cases, the absence of 5-hydroxymethylcytosine (5hmC) was observed. We present seven cases of ALK+ LBCL, discussing the correlation between fusion genes and ALK protein expression, and enhancing our understanding of the genetic attributes of this tumour. This study also shows the loss of 5hmC in nearly all seven ALK+ LBCL cases, independently of TET2 mutations.

RESUMEN

BACKGROUND: TFE3-rearranged renal cell carcinoma (TFE3-rRCC) is a rare but highly heterogeneous renal cell carcinoma (RCC) entity, of which the clinical treatment landscape is largely undefined. This study aims to evaluate and compare the efficacy of different systemic treatments and further explore the molecular correlates. METHODS: Thirty-eight patients with metastatic TFE3-rRCC were enrolled. Main outcomes included progression-free survival (PFS), overall survival, objective response rate (ORR) and disease control rate. RNA sequencing was performed on 32 tumors. RESULTS: Patients receiving first-line immune checkpoint inhibitor (ICI) based combination therapy achieved longer PFS than those treated without ICI (median PFS: 11.5 vs. 5.1 months, P = 0.098). After stratification of fusion partners, the superior efficacy of first-line ICI based combination therapy was predominantly observed in ASPSCR1-TFE3 rRCC (median PFS: not reached vs. 6.5 months, P = 0.01; ORR: 67.5% vs. 10.0%, P = 0.019), but almost not in non-ASPSCR1-TFE3 rRCC. Transcriptomic data revealed enrichment of ECM and collagen-related signaling in ASPSCR1-TFE3 rRCC, which might interfere with the potential efficacy of anti-angiogenic monotherapy. Whereas angiogenesis and immune activities were exclusively enriched in ASPSCR1-TFE3 rRCC and promised the better clinical outcomes with ICI plus tyrosine kinase inhibitor combination therapy. CONCLUSIONS: The current study represents the largest cohort comparing treatment outcomes and investigating molecular correlates of metastatic TFE3-rRCC based on fusion partner stratification. ICI based combination therapy could serve as an effective first-line treatment option for metastatic ASPSCR1-TFE3 rRCC patients. Regarding with other fusion subtypes, further investigations should be performed to explore the molecular mechanisms to propose pointed therapeutic strategy accordingly.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Carcinoma de Células Renales , Inhibidores de Puntos de Control Inmunológico , Neoplasias Renales , Proteínas de Fusión Oncogénica , Humanos , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/mortalidad , Femenino , Masculino , Persona de Mediana Edad , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Neoplasias Renales/mortalidad , Anciano , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Proteínas de Fusión Oncogénica/genética , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Reordenamiento Génico , Biomarcadores de Tumor/genética , Resultado del Tratamiento , Pronóstico , Péptidos y Proteínas de Señalización Intracelular/genética

RESUMEN

BACKGROUND: Non-small cell lung cancer (NSCLC) harboring ROS1 rearrangements is a molecular subset that exhibits favorable responses to tyrosine kinase inhibitor (TKI) treatment than chemotherapy. This study investigated real-world treatment patterns and survival outcomes among patients with ROS1-rearranged advanced NSCLC. METHODS: We conducted a retrospective analysis of patients with ROS1-rearranged advanced NSCLC treated in four different hospitals in China from August 2018 to March 2022. The study analyzed gene fusion distribution, resistance patterns, and survival outcomes. RESULTS: ROS1 rearrangement occurs in 1.8 % (550/31,225) of our study cohort. CD74 was the most common ROS1 fusion partner, accounting for 45.8 %. Crizotinib was used in 73.9 % of patients in the first-line treatment, and an increased use of chemotherapy, ceritinib, and lorlatinib was seen in the second-line setting. Lung (43.2 %) and brain (27.6 %) were the most common sites of progression in first-line setting, while brain progression (39.2 %) was the most common site of progression in second-line. Median overall survival was 46 months (95 % confidence intervals: 39.6-52.4). First-line crizotinib use yielded significantly superior survival outcomes over chemotherapy in terms of progression-free (18.5 vs. 6.0; p < 0.001) and overall survival (49.8 vs. 37; p = 0.024). The choice of treatment in the latter line also had survival implications, wherein survival outcomes were better when first-line crizotinib was followed by sequential TKI therapy than first-line chemotherapy followed by TKI therapy. CONCLUSIONS: Our study provided insights into the real-world treatment, drug resistance patterns, and survival outcomes among patients with ROS1-rearranged NSCLC. This information serves as a valuable reference for guiding the treatment of this molecular subset of NSCLC.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Crizotinib , Reordenamiento Génico , Neoplasias Pulmonares , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas , Humanos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Estudios Retrospectivos , Masculino , Proteínas Proto-Oncogénicas/genética , Femenino , Proteínas Tirosina Quinasas/genética , Persona de Mediana Edad , Anciano , Crizotinib/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Adulto , Tasa de Supervivencia , Pronóstico , Resistencia a Antineoplásicos/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Anciano de 80 o más Años , Pirazoles/uso terapéutico , China/epidemiología , Aminopiridinas , Antígenos de Diferenciación de Linfocitos B , Antígenos de Histocompatibilidad Clase II , Lactamas

RESUMEN

INTRODUCTION: Variable partners and breakpoints have been reported in patients with ROS1-rearranged NSCLC. Here, we investigated the association of fusion partners and breakpoints with crizotinib efficacy in NSCLCs with common ROS1 fusions. METHODS: DNA and RNA next-generation sequencing (NGS) and immunohistochemistry were performed to characterize ROS1 fusions. RESULTS: Using DNA NGS, we identified ROS1 fusions in 210 cases, comprising 171 common (CD74/EZR/TPM3/SDC4/SLC34A2-ROS1) and 39 uncommon (variants identified in <5%) ROS1 fusion cases. DNA NGS detected variable ROS1 genomic breakpoints in common ROS1 fusions, whereas RNA NGS found ROS1 breakpoints mainly occurring in exons 32, 34 and 35, resulting in long (exon 32) and short (exon 34 or 35) ROS1 fusions. ROS1 immunohistochemistry revealed that membranous and cytoplasmic staining was predominant in long ROS1 fusions, whereas cytoplasmic staining was predominant in short ROS1 fusions (p = 0.006). For patients who received first-line crizotinib, median progression-free survival (mPFS) was lower in patients with long ROS1 fusions than those with short ROS1 fusions (8.0 versus 24.0 mo, p = 0.006). Moreover, mPFS for patients with and without TP53 mutations was 8.0 and 19.0 months, respectively (p = 0.159); mPFS for patients with and without BIM deletion polymorphism was 5.0 and 22.0 months, respectively (p = 0.003). When analyzing together with fusion partners, patients with long CD74/SLC34A2-ROS1 fusions were found to have shorter PFS than those with other ROS1, regardless of the presence or absence of TP53 mutations (p < 0.001 and p = 0.002, respectively). CONCLUSIONS: Long CD74/SLC34A2-ROS1 fusions, which retain transmembrane regions in ROS1 and fusion partners, are associated with poor response to crizotinib independent of TP53 mutations.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Antígenos de Histocompatibilidad Clase II , Neoplasias Pulmonares , Proteínas de Fusión Oncogénica , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Crizotinib/farmacología , Crizotinib/uso terapéutico , ADN , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Proteínas de Fusión Oncogénica/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIb/genética , Proteína p53 Supresora de Tumor/genética , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Diferenciación de Linfocitos B/genética

RESUMEN

Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

RESUMEN

Zinc finger protein 384 (ZNF384) encodes a C2H2-type zinc finger protein that can function as a transcription factor. ZNF384 rearrangement in acute lymphoblastic leukemia (ALL) was first reported in 2002. More than 19 different ZNF384 fusion partners have been detected in ALL. These include E1A-binding protein P300 (EP300), CREB-binding protein (CREBBP), transcription factor 3 (TCF3), TATA-box binding protein associated factor 15 (TAF15), Ewing sarcoma breakpoint region 1 gene (EWSR1), AT-rich interactive domain-containing protein 1B (ARID1B), SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 2 (SMARCA2), synergin gamma (SYNRG), clathrin heavy chain (CLTC), bone morphogenic protein 2-inducible kinase (BMP2K), Nipped-B-like protein (NIPBL), A Kinase Anchoring Protein 8 (AKAP8), Chromosome 11 Open Reading Frame 74 (C11orf74), DEAD-Box Helicase 42 (DDX42), ATP Synthase F1 Subunit Gamma (ATP2C1), Euchromatic Histone Lysine Methyltransferase 1 (EHMT1), Testic Expressed 41 (TEX41), etc. Patients diagnosed with ALL harboring ZNF384 rearrangements commonly had a good prognosis. The mechanisms, performance, and features of different ZNF384 rearrangements in acute lymphoblastic leukemia have been well evaluated.

Asunto(s)

Actinas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Cromatina , Proteínas de Ciclo Celular , ADN Helicasas , Proteínas Nucleares , Factores de Transcripción , Transactivadores , ATPasas Transportadoras de Calcio

RESUMEN

In the fermentative production of compounds by using microorganisms, control of the transporter activity responsible for substrate uptake and product efflux, in addition to intracellular metabolic modification, is important from a productivity perspective. However, there has been little progress in analyses of the functions of microbial membrane transporters, and because of the difficulty in finding transporters that transport target compounds, only a few transporters have been put to practical use. Here, we constructed a Corynebacterium glutamicum-derived transporter expression library (CgTP-Express library) with the fusion partner gene mstX and used a peptide-feeding method with the dipeptide L-Ala-L-Ala to search for alanine exporters in the library. Among 39 genes in the library, five candidate alanine exporters (NCgl2533, NCgl2683, NCgl0986, NCgl0453, and NCgl0929) were found; expression of NCgl2533 increased the alanine concentration in cell culture. The CgTP-Express library was thus effective for finding a new transporter candidate.

Asunto(s)

Corynebacterium glutamicum , Proteínas de Transporte de Membrana , Fermentación , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Alanina/genética , Alanina/metabolismo , Transporte Biológico , Ingeniería Metabólica/métodos

RESUMEN

Targeting molecular alterations has been proven to be an inflecting point in tumor treatment. Especially in recent years, inhibitors that target the tyrosine receptor kinase show excellent response rates and durable effects in all kind of tumors that harbor fusions of one of the three neurotrophic tyrosine receptor kinase genes (NTRK1, NTRK2 and NTRK3). Today, the therapeutic options in most metastatic sarcomas are rather limited. Therefore, identifying which sarcoma types are more likely to harbor these targetable NTRK fusions is of paramount importance. At the moment, identification of these fusions is solely based on immunohistochemistry and confirmed by molecular techniques. However, a first attempt has been made to describe the histomorphology of NTRK-fusion positive sarcomas, in order to pinpoint which of these tumors are the best candidates for testing. In this study, we investigate the immunohistochemical expression of pan-TRK in 70 soft tissue and bone sarcomas. The pan-TRK positive cases were further investigated with molecular techniques for the presence of a NTRK fusion. Seven out of the 70 cases showed positivity for pan-TRK, whereas two of these seven cases presented an NTRK3 fusion. Further analysis of the fused sarcomas revealed some unique histological, molecular and clinical findings. The goal of this study is to expand the histomorphological spectrum of the NTRK-fused sarcomas, to identify their fusion partners and to correlate these parameters with the clinical outcome of the disease. In addition, we evaluated the immunohistochemical expression pattern of the pan-TRK and its correlation with the involved NTRK gene.

Asunto(s)

Sarcoma , Neoplasias de los Tejidos Blandos , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/genética , Humanos , Patología Molecular , Receptor trkA/genética , Sarcoma/genética , Neoplasias de los Tejidos Blandos/genética

RESUMEN

Epithelioid fibrous histiocytoma (EFH) is a rare cutaneous neoplasm, which is characterized by the presence of rearrangements involving the ALK gene. Although EFH was long considered a variant of fibrous histiocytoma, the identification of its unique genetic signature confirmed that it represents a distinct entity. The aim of the present study was to examine a cohort of ALK-immunoreactive EFH cases to further characterize gene fusion partners. Next generation sequencing detected ALK fusions in 11 EFH cases identified in the pathology archives of two different institutions. The most common fusion partner was DCTN1 (N = 4) followed by CLTC (N = 2) and VCL (N = 2), while the remaining cases harbored fusions involving SPECC1L, PPFIBP1, and PRKAR1A. In one case no fusion was detected by NGS and FISH despite suitable sample quality. Notably, IHC demonstrated positive ALK expression and the level of aligned ALK reads was comparable to the fusion-positive cases. To the best of our knowledge, this is the first report of CLTC as a fusion partner in EFH. The two CLTC rearranged cases in our cohort also represent the first two EFH cases in the literature that involve exon 19 of ALK, instead of exon 20. These findings underscore the remarkable plasticity of ALK as an oncogenic driver and further expand the list of its potential fusion partners in EFH. Lastly this is also the first report of ALK-immunoreactive EFH with no underlying fusion suggesting a fusion independent transcription mechanism as seen in other tumors.

Asunto(s)

Cadenas Pesadas de Clatrina , Histiocitoma Fibroso Benigno , Neoplasias Cutáneas , Quinasa de Linfoma Anaplásico/genética , Cadenas Pesadas de Clatrina/genética , Fusión Génica , Histiocitoma Fibroso Benigno/genética , Histiocitoma Fibroso Benigno/metabolismo , Histiocitoma Fibroso Benigno/patología , Humanos , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias Cutáneas/genética , Activación Transcripcional

RESUMEN

For the efficient production of heterologous proteins in the yeast Saccharomyces cerevisiae, we screened for a novel fusion partner from the yeast secretome. From twenty major proteins identified from the yeast secretome, we selected Scw4p, a cell wall protein with similarity to glucanase, and modified to develop a general fusion partner for the secretory expression of heterologous proteins in yeast. The optimal size of the SCW4 gene to act as an efficient fusion partner was determined by C-terminal truncation analysis; two of the variants, S1 (truncated at codon 115Q) and S2 (truncated at codon 142E), were further used for the secretion of heterologous proteins. When fused with S2, the secretion of three target proteins (hGH, exendin-4, and hPTH) significantly increased. Conserved O-glycosylation sites (Ser/Thr-rich domain) and hydrophilic sequences of S2 were deemed important for the function of S2 as a secretion fusion partner. Approximately 5 g/L of the S2-exendin-4 fusion protein was obtained from fed-batch fermentation. Intact target proteins were easily purified by affinity chromatography after in vitro processing of the fusion partner. This system may be of general application for the secretory production of heterologous proteins in S. cerevisiae. KEY POINTS : • Target proteins were efficiently secreted with their N-terminus fused to Scw4p. • O-glycosylation and hydrophilic stretches in Scw4p were important for protein secretion. • A variant of Scw4p (S2) was successfully applied for the secretory expression of heterologous proteins.

Asunto(s)

Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Secretoma

RESUMEN

G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A2A receptor (A2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A2AR through straight helical connections without any kinks or intervening loops. The chimeric A2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.

Asunto(s)

Agonistas del Receptor de Adenosina A2/metabolismo , Proteínas/metabolismo , Receptor de Adenosina A2A/química , Receptor de Adenosina A2A/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Adenosina/metabolismo , Agonistas del Receptor de Adenosina A2/química , Humanos , Ligandos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica en Hélice alfa , Proteínas/química , Proteínas Recombinantes de Fusión/química

RESUMEN

Malignant gastrointestinal neuroectodermal tumor (GNET) is a rare malignant primary gastrointestinal mesenchymal tumor which can be diagnosed via fine-needle aspiration (FNA) cytology. In the context of FNA, the diagnosis requires a cell block and the use of significant resources including immunohistochemical stains and molecular testing. The differential diagnosis of GNET includes clear cell sarcoma (CCS), gastrointestinal stromal tumor (GIST), gastric schwannoma, metastatic melanoma, malignant perivascular epithelioid cell tumor (PEComa) and granular cell tumor, among others. Here we describe a case which was initially diagnosed as malignant granular cell tumor by FNA which was later revised to GNET following the finding of an EWSR1-ATF1 fusion gene rearrangement.

Asunto(s)

Tracto Gastrointestinal/patología , Tumores Neuroectodérmicos , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Biopsia con Aguja Fina , Proteínas de Unión a Calmodulina/análisis , Proteínas de Unión a Calmodulina/metabolismo , Diagnóstico Diferencial , Femenino , Neoplasias Gastrointestinales/diagnóstico , Neoplasias Gastrointestinales/metabolismo , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/diagnóstico , Tumores del Estroma Gastrointestinal/metabolismo , Tumores del Estroma Gastrointestinal/patología , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Melanoma/diagnóstico , Melanoma/metabolismo , Melanoma/patología , Persona de Mediana Edad , Tumores Neuroectodérmicos/diagnóstico , Tumores Neuroectodérmicos/metabolismo , Tumores Neuroectodérmicos/patología , Sarcoma de Células Claras/diagnóstico , Sarcoma de Células Claras/metabolismo , Sarcoma de Células Claras/patología

RESUMEN

Soybean seeds are an ideal host for the production of recombinant proteins because of their high content of proteins, long-term stability of seed proteins under ambient conditions, and easy establishment of efficient purification protocols. In this study, a polypeptide fusion strategy was applied to explore the capacity of elastin-like polypeptide (ELP) and γ-zein fusions in increasing the accumulation of the recombinant protein in soybean seeds. Transgenic soybean plants were generated to express the γ-zein- or ELP-fused green fluorescent protein (GFP) under the control of the soybean seed-specific promoter of ß-conglycinin alpha subunit (BCSP). Significant differences were observed in the accumulation of zein-GFP and GFP-ELP from that of the unfused GFP in transgenic soybean seeds based on the total soluble protein (TSP), despite the low-copy of T-DNA insertions and similar expression at the mRNA levels in selected transgenic lines. The average levels of zein-GFP and GFP-ELP accumulated in immature seeds of these transgenic lines were 0.99% and 0.29% TSP, respectively, compared with 0.07% TSP of the unfused GFP. In mature soybean seeds, the accumulation of zein-GFP and GFP-ELP proteins was 1.8% and 0.84% TSP, an increase of 3.91- and 1.82-fold, respectively, in comparison with that of the unfused GFP (0.46% TSP). Confocal laser scanning analysis showed that both zein-GFP and GFP-ELP were abundantly deposited in many small spherical particles of transgenic seeds, while there were fewer such florescence signals in the same cellular compartments of the unfused GFP-expressing seeds. Despite increased recombinant protein accumulation, there were no significant changes in the total protein and oil content in seeds between the transgenic and non-transformed plants, suggesting the possible presence of threshold limits of total protein accumulation in transgenic soybean seeds. Overall, our results indicate that γ-zein and ELP fusions significantly increased the accumulation of the recombinant protein, but exhibited no significant influence on the total protein and oil content in soybean seeds.

Asunto(s)

Glycine max , Zeína , Elastina/genética , Péptidos , Plantas Modificadas Genéticamente/genética , Proteínas Recombinantes de Fusión/genética , Semillas/genética , Glycine max/genética , Zeína/genética

RESUMEN

Hydrophobins are small amphipathic proteins excreted from filamentous fungi that self-assemble into the amphipathic film at hydrophobic/hydrophilic interfaces and can be used in a wide range of biotechnological application such as antimicrobial coatings, biosensors, and drug delivery. Here we describe a simple method for producing functionally active class I and class II hydrophobins in E. coli. The class I hydrophobin EAS (rodlet protein) from Neurospora crassa and class II hydrophobin HFBII from Trichoderma reesei were separately fused with fusion partner Ffu312 (ß-fructofuranosidase truncation with a native signal peptide) and successfully expressed in E. coli. Significantly, fused hydrophobins Ffu312-EAS and Ffu312-HFBII were excreted into the culture medium. The excretory expression of hydrophobins facilitated the correct disulfide-bond formation and simplified the purification. Both fusion hydrophobins reversed the glass surface hydrophilicity, reduced the water surface tension and improved emulsion stability. Ffu312 has little effect on surface coating, water surface tension and emulsion stabilization of hydrophobins. This study may provide an efficient approach for excretory and functional expression of class I and class II hydrophobins in E. coli.

Asunto(s)

Proteínas Fúngicas/biosíntesis , Interacciones Hidrofóbicas e Hidrofílicas/efectos de los fármacos , Tensoactivos/química , Antiinfecciosos/química , Técnicas Biosensibles , Sistemas de Liberación de Medicamentos , Escherichia coli/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/farmacología , Hypocreales/química , Hypocreales/genética , Neurospora crassa/química , Neurospora crassa/genética , Propiedades de Superficie/efectos de los fármacos , Tensión Superficial/efectos de los fármacos , Agua/química

RESUMEN

Steviol glycosides (SGs) with zero calories and high-intensity sweetness are the best substitutes of sugar for the human diet. Uridine diphosphate dependent glycosyltransferase (UGT) UGT76G1, as a key enzyme for the biosynthesis of SGs with a low heterologous expression level, hinders its application. In this study, a suitable fusion partner, Smt3, was found to enhance the soluble expression of UGT76G1 by 60%. Additionally, a novel strategy to improve the expression of Smt3-UGT76G1 was performed, which co-expressed endogenous genes prpD and malK in Escherichia coli. Notably, this is the first report of constructing an efficient E. coli expression system by regulating prpD and malK expression, which remarkably improved the expression of Smt3-UGT76G1 by 200% as a consequence. Using the high-expression strain E. coli BL21 (DE3) M/P-3-S32U produced 1.97 g/L of Smt3-UGT76G1 with a yield rate of 61.6 mg/L/h by fed-batch fermentation in a 10 L fermenter. The final yield of rebadioside A (Reb A) and rebadioside M (Reb M) reached 4.8 g/L and 1.8 g/L, respectively, when catalyzed by Smt3-UGT76G1 in the practical UDP-glucose regeneration transformation system in vitro. This study not only carried out low-cost biotransformation of SGs but also provided a novel strategy for improving expression of heterologous proteins in E. coli.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Glicósidos/biosíntesis , Glicosiltransferasas/metabolismo , Hidroliasas/metabolismo , Biocatálisis , Reactores Biológicos/microbiología , Biotransformación , Fermentación , Glicósidos/química , Glicosilación , Plásmidos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética/genética , Solubilidad

RESUMEN

Brevibacillus offers great potential as a recombinant protein expression host because of its exceptional abilities to synthesize and excrete proteins and its low extracellular protease activity. Despite these strengths, effective recombinant expression strategies are still the key to achieving high-level expression of recombinant proteins in Brevibacillus due to individual differences among strains and target proteins. Many strategies have been developed to improve recombinant protein expression in Brevibacillus. This review begins by introducing the processes used to establish and apply the Brevibacillus expression system, and then critically discusses the strategies available for improving recombinant protein expression in Brevibacillus, including optimization of the host and the expression vector, co-expression of a fusion partner or foldase, and optimization of the fermentation process. Finally, the prospects for further improvement of recombinant protein expression based on Brevibacillus are also discussed. This review is intended to provide a strategic reference for scientists wanting to improve the expression of a specific recombinant protein in Brevibacillus or other expression systems.

Asunto(s)

Brevibacillus , Ingeniería Genética , Proteínas Recombinantes de Fusión , Brevibacillus/genética , Brevibacillus/metabolismo , Clonación Molecular , Fermentación , Vectores Genéticos , Plásmidos , Regiones Promotoras Genéticas

RESUMEN

BACKGROUND: Insulin is a peptide hormone used for regulating blood glucose levels. Human insulin market is projected to grow at a rate of 12.5% annually. To meet the needs of patients, a cost effective insulin manufacturing strategy has to be developed. This can be achieved by selecting a competent host, ideal fusion tag and streamlined downstream process. OBJECTIVE: In this article, we have demonstrated that selecting a right fusion partner for expression of toxic proteins like insulin, plays a major role in increasing the recombinant protein yield. METHODS: In this article, we have focused on identifying a peptide tag fusion partner for expressing proinsulin by truncating thioredoxin tag. Truncations were carried out from both Amino and Carboxy terminus of the protein and efficiency of truncated sequences was evaluated by expressing it with proinsulin gene. FCTRX (1-15) sequence fused to proinsulin was processed further to establish downstream protocol for purification. RESULTS: Thioredoxin tag was truncated appropriately by considering the fusion tag: protein ratio. A couple of sequences ranging 10 - 15 amino acids were identified based on its in silico properties. Of these FCTRX (1-15) showed increased expression and stability of fusion protein. 156 mg of purified insulin was generated from 1g of inclusion body after enzymatic conversion and chromatographic steps. CONCLUSION: As a result of the current study, it was concluded that FCTRX (1-15) peptide has advantageous attributes to be considered as an ideal fusion tag for expression of proinsulin. This can be further explored by expressing it with other proteins.

Asunto(s)

Proinsulina/química , Proinsulina/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Tiorredoxinas/química , Tiorredoxinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Glucemia/metabolismo , Cromatografía Liquida , Clonación Molecular , Enteropeptidasa/metabolismo , Escherichia coli/genética , Regulación de la Expresión Génica , Humanos , Cuerpos de Inclusión/metabolismo , Pliegue de Proteína , Solubilidad

RESUMEN

ROS1 fusion-positive (ROS1+) NSCLC was discovered in 2007, the same year as the discovery of ALK-positive (ALK+) NSCLC but has trailed ALK+ NSCLC in terms of development. There seems to be a differential response to ROS1 inhibitors, which depend on fusion partners (CD74, SLC34A2, or SDC4); thus, knowledge of the fusion partners in ROS1+ NSCLC is important. To date (end of February 2020), we have identified 24 unique 5' fusion partners of ROS1 in ROS1+ NSCLC from published literature and congress proceedings. Thus, we published this catalog for easy reference.

RESUMEN

The Escherichia coli (E. coli) expression system has been widely used to produce recombinant proteins. However, in some heterologous expressions, there are still difficulties in large-scale production. The use of fusion partners is one of the strategies for improving the expression levels of proteins in E. coli host. Here, we demonstrate a novel fusion element, the NT11-tag, which enhances protein expression. The NT11-tag was derived from the first 11 amino acid residues within the N-terminal N-half domain of a duplicated carbonic anhydrase (dCA) from Dunaliella species. Previously, we have found that the tag improves expression of the C-half domain of dCA when linked to its N-terminus. To verify its use as a protein production enhancer tag, two kinds of CAs derived from Hahella chejuensis (Hc-CA) and Thermovibrio ammonifican (Ta-CA) and the yellow fluorescent protein (YFP) were used as model proteins to measure their increased expression upon fusion with the NT11-tag. The NT11-tag amplified protein expression in E. coli by 6.9- and 7.6-fold for Ta-CA and YFP, respectively. Moreover, the tag also enhanced the soluble expression of Hc-CA, Ta-CA, and YFP by 1.7-, 5.0-, and 3.2-fold, respectively. Furthermore, protein yield was increased without inhibiting protein function. These results indicate that the use of the NT11-tag is a promising method for improving protein production in E. coli.

Asunto(s)

Proteínas Bacterianas/biosíntesis , Proteínas de Escherichia coli/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica/genética , Proteínas Luminiscentes/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Bacterianas/genética , Anhidrasas Carbónicas/genética , Proteínas de Escherichia coli/genética , Proteínas Luminiscentes/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/genética