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1.
Metab Eng ; 72: 171-187, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35301123

RESUMO

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.


Assuntos
Via Secretória , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Proteínas Recombinantes , Via Secretória/genética
2.
J Biol Chem ; 293(15): 5492-5508, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29414779

RESUMO

Neurturin (NRTN) provides trophic support to neurons and is considered a therapeutic agent for neurodegenerative diseases, such as Parkinson's disease. It binds to its co-receptor GFRa2, and the resulting NRTN-GFRa2 complex activates the transmembrane receptors rearranged during transfection (RET) or the neural cell adhesion molecule (NCAM). We report the crystal structure of NRTN, alone and in complex with GFRa2. This is the first crystal structure of a GFRa with all three domains and shows that domain 1 does not interact directly with NRTN, but it may support an interaction with RET and/or NCAM, via a highly conserved surface. In addition, biophysical results show that the relative concentration of GFRa2 on cell surfaces can affect the functional affinity of NRTN through avidity effects. We have identified a heparan sulfate-binding site on NRTN and a putative binding site in GFRa2, suggesting that heparan sulfate has a role in the assembly of the signaling complex. We further show that mutant NRTN with reduced affinity for heparan sulfate may provide a route forward for delivery of NRTN with increased exposure in preclinical in vivo models and ultimately to Parkinson's patients.


Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Heparitina Sulfato/química , Complexos Multiproteicos/química , Neurturina/química , Transdução de Sinais , Cristalografia por Raios X , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neurturina/genética , Neurturina/metabolismo , Domínios Proteicos , Estrutura Quaternária de Proteína
3.
Biotechnol Bioeng ; 114(10): 2348-2359, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28627739

RESUMO

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc.


Assuntos
Melhoramento Genético/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Inibidor Tecidual de Metaloproteinase-2/biossíntese , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Proliferação de Células/fisiologia , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Proteínas Recombinantes/isolamento & purificação , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-3/genética
4.
Methods Mol Biol ; 2810: 75-83, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38926273

RESUMO

Large culture volumes are often required when expression constructs are particularly low-yielding or when end uses require significant amounts of material. In these cases, a single homogeneous culture is usually more convenient, in terms of both consistency of expression and labor/resource requirements, than multiple parallel cultures. Using a WAVE Bioreactor culture, volumes as high as 500L may be achieved in a single vessel. Here, we describe the transfection of Expi293F cells in a disposable 50L Cellbag on a WAVE Bioreactor platform to produce recombinant protein. The methods described herein may be adapted, with suitable optimizations, for other suspension-adapted mammalian cell lines.


Assuntos
Reatores Biológicos , Proteínas Recombinantes , Transfecção , Transfecção/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Humanos , Animais , Linhagem Celular , Técnicas de Cultura de Células/métodos , Expressão Gênica
5.
Clin Transl Immunology ; 10(7): e1312, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34295471

RESUMO

OBJECTIVE: The COVID-19 pandemic poses an immense need for accurate, sensitive and high-throughput clinical tests, and serological assays are needed for both overarching epidemiological studies and evaluating vaccines. Here, we present the development and validation of a high-throughput multiplex bead-based serological assay. METHODS: More than 100 representations of SARS-CoV-2 proteins were included for initial evaluation, including antigens produced in bacterial and mammalian hosts as well as synthetic peptides. The five best-performing antigens, three representing the spike glycoprotein and two representing the nucleocapsid protein, were further evaluated for detection of IgG antibodies in samples from 331 COVID-19 patients and convalescents, and in 2090 negative controls sampled before 2020. RESULTS: Three antigens were finally selected, represented by a soluble trimeric form and the S1-domain of the spike glycoprotein as well as by the C-terminal domain of the nucleocapsid. The sensitivity for these three antigens individually was found to be 99.7%, 99.1% and 99.7%, and the specificity was found to be 98.1%, 98.7% and 95.7%. The best assay performance was although achieved when utilising two antigens in combination, enabling a sensitivity of up to 99.7% combined with a specificity of 100%. Requiring any two of the three antigens resulted in a sensitivity of 99.7% and a specificity of 99.4%. CONCLUSION: These observations demonstrate that a serological test based on a combination of several SARS-CoV-2 antigens enables a highly specific and sensitive multiplex serological COVID-19 assay.

6.
ACS Synth Biol ; 8(9): 1998-2006, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31398008

RESUMO

The ability to manipulate the expression of mammalian genes using synthetic transcription factors is highly desirable in both fields of basic research and industry for diverse applications, including stem cell reprogramming and differentiation, tissue engineering, and drug discovery. Orthogonal CRISPR systems can be used for simultaneous transcriptional upregulation of a subset of target genes while downregulating another subset, thus gaining control of gene regulatory networks, signaling pathways, and cellular processes whose activity depends on the expression of multiple genes. We have used a rapid and efficient modular cloning system to build and test in parallel diverse CRISPRa and CRISPRi systems and develop an efficient orthogonal gene regulation system for multiplexed and simultaneous up- and downregulation of endogenous target genes.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Reprogramação Celular , Regulação da Expressão Gênica , Redes Reguladoras de Genes/genética , Células HCT116 , Humanos , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Transdução de Sinais/genética , Engenharia Tecidual
7.
FEBS Lett ; 592(14): 2499-2511, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29933498

RESUMO

Due in part to the needs of the biopharmaceutical industry, there has been an increased drive to generate high quality recombinant proteins in large amounts. However, achieving high yields can be a challenge as the novelty and increased complexity of new targets often makes them 'difficult-to-express'. This study aimed to define the molecular features that restrict the production of a model 'difficult-to-express' recombinant protein, Tissue Inhibitor Metalloproteinase-3 (TIMP-3). Building from experimental data, computational approaches were used to rationalize the redesign of this recombinant target to generate a chimera with enhanced secretion. The results highlight the importance of early identification of unfavourable sequence attributes, enabling the generation of engineered protein forms that bypass 'secretory' bottlenecks and result in efficient recombinant protein production.


Assuntos
Clonagem Molecular/métodos , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Biologia Computacional , Cricetinae , Cricetulus , Expressão Gênica , Humanos , Camundongos , Modelos Biológicos , Transporte Proteico/genética , Proteínas Recombinantes/química , Via Secretória/genética , Biologia Sintética/métodos , Inibidor Tecidual de Metaloproteinase-2/química , Inibidor Tecidual de Metaloproteinase-2/genética , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Inibidor Tecidual de Metaloproteinase-3/química , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo
8.
ACS Med Chem Lett ; 9(7): 600-605, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30034586

RESUMO

A lead generation campaign identified indole-based sPLA2-X inhibitors with a promising selectivity profile against other sPLA2 isoforms. Further optimization of sPLA2 selectivity and metabolic stability resulted in the design of (-)-17, a novel, potent, and selective sPLA2-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (-)-17 was tested in an ApoE-/- murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA2-X inhibition on atherosclerosis development. Despite being well tolerated and achieving adequate systemic exposure of mechanistic relevance, (-)-17 did not significantly affect circulating lipid and lipoprotein biomarkers and had no effect on coronary function or histological markers of atherosclerosis.

9.
ACS Med Chem Lett ; 9(7): 594-599, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30034585

RESUMO

In order to assess the potential of sPLA2-X as a therapeutic target for atherosclerosis, novel sPLA2 inhibitors with improved type X selectivity are required. To achieve the objective of identifying such compounds, we embarked on a lead generation effort that resulted in the identification of a novel series of indole-2-carboxamides as selective sPLA2-X inhibitors with excellent potential for further optimization.

10.
PLoS One ; 9(8): e103774, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25105596

RESUMO

Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.


Assuntos
Caspases/fisiologia , Linfócitos/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/fisiologia , Transdução de Sinais/fisiologia , Transcrição Gênica/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína 10 de Linfoma CCL de Células B , Sequência de Bases , Western Blotting , Caspases/genética , Caspases/metabolismo , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Células Jurkat , Dados de Sequência Molecular , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Mutação de Sentido Incorreto/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteólise , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Antígenos/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/genética , Transcrição Gênica/fisiologia
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