An improved method for the heterologous production of soluble human ribosomal proteins in Escherichia coli

Correddu, Danilo; López, José de Jesús Montaño; Vadakkedath, Praveen G.; Lai, Amy; Pernes, Jane I.; Watson, Paris R.; Leung, Ivanhoe K. H.

doi:10.1038/s41598-019-45323-8

Cited by 13 publications

(5 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Because Trx1 is an essential regulator of cell metabolism 39 , 40 , we identified Trx1 direct interacting proteins involved in citrate cycle and amino acid metabolism. Trx1 can also stabilize ribosomal proteins 41 , 42 . As expected, ribosomal proteins were significantly enriched in our Trx1 cross-linking protein dataset.…”

Section: Resultsmentioning

confidence: 99%

Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids

Liu,

Ding,

Cheng

et al. 2024

Nat Commun

View full text Add to dashboard Cite

Latent bioreactive unnatural amino acids (Uaas) have been widely used in the development of covalent drugs and identification of protein interactors, such as proteins, DNA, RNA and carbohydrates. However, it is challenging to perform high-throughput identification of Uaa cross-linking products due to the complexities of protein samples and the data analysis processes. Enrichable Uaas can effectively reduce the complexities of protein samples and simplify data analysis, but few cross-linked peptides were identified from mammalian cell samples with these Uaas. Here we develop an enrichable and multiple amino acids reactive Uaa, eFSY, and demonstrate that eFSY is MS cleavable when eFSY-Lys and eFSY-His are the cross-linking products. An identification software, AixUaa is developed to decipher eFSY mass cleavable data. We systematically identify direct interactomes of Thioredoxin 1 (Trx1) and Selenoprotein M (SELM) with eFSY and AixUaa.

show abstract

Section: Resultsmentioning

confidence: 99%

Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids

Liu,

Ding,

Cheng

et al. 2024

Nat Commun

View full text Add to dashboard Cite

show abstract

“…MSOX catalyzes the oxidation of sarcosine to glycine and formaldehyde giving H is known that for the heterologous expression of some proteins, the gene induction at low temperature is particularly useful when the protein is readily degraded by proteases, aggregates, or is conveyed into inclusion bodies. [43] Moreover, a key point to produce a high amount of soluble CYP116B5-fl was the fine adjustment of gene expression. For this matter, different concentrations of IPTG (from 0 to 1 mM) were tested.…”

Section: Discussionmentioning

confidence: 99%

Catalytically self‐sufficient CYP116B5: Domain switch for improved peroxygenase activity

Correddu

Catucci

Giuriato

et al. 2023

Biotechnology Journal

View full text Add to dashboard Cite

Self‐sufficient cytochromes P450 of the sub‐family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions toward a wide range of organic compounds. However, these P450s are often unstable in solution and their activity is limited to a short reaction time. Previously it has been shown that the isolated heme domain of CYP116B5 can work as a peroxygenase with H2O2 without the addition of NAD(P)H. In this work, protein engineering was used to generate a chimeric enzyme (CYP116B5‐SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) capable of producing H2O2. The full‐length enzyme (CYP116B5‐fl) is characterized for the first time, allowing a detailed comparison to the heme domain (CYP116B5‐hd) and CYP116B5‐SOX. The catalytic activity of the three forms of the enzyme was studied using p‐nitrophenol as substrate, and adding NADPH (CYP116B5‐fl), H2O2 (CYP116B5‐hd), and sarcosine (CYP116B5‐SOX) as source of electrons. CYP116B5‐SOX performs better than CYP116B5‐fl and CYP116B5‐hd showing 10‐ and 3‐folds higher activity, in terms of p‐nitrocatechol produced per mg of enzyme per minute. CYP116B5‐SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.

show abstract

“…The rAMEV2 construct consists of A. baumannii thioredoxin (TrxA) protein linked to five predicted immunogenic peptides (Table 1). Although TrxA is a well-characterized virulence factor, it also assists in improving the solubility of the rAMEV2 construct [31][32][33][34]. Peptides were joined by either GPGPG or KK linkers to limit the creation of irrelevant pseudo epitopes [35][36][37].…”

Section: Generation Of An Acinetobacter Multi-epitope Vaccine Amev2mentioning

confidence: 99%

Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection

Jeffreys,

Tompkins,

Aki

et al. 2024

Vaccines

View full text Add to dashboard Cite

Multi-drug-resistant (MDR) Acinetobacter baumannii is an opportunistic pathogen associated with hospital-acquired infections. Due to its environmental persistence, virulence, and limited treatment options, this organism causes both increased patient mortality and incurred healthcare costs. Thus, prophylactic vaccination could be ideal for intervention against MDR Acinetobacter infection in susceptible populations. In this study, we employed immunoinformatics to identify peptides containing both putative B- and T-cell epitopes from proteins associated with A. baumannii pathogenesis. A novel Acinetobacter Multi-Epitope Vaccine (AMEV2) was constructed using an A. baumannii thioredoxin A (TrxA) leading protein sequence followed by five identified peptide antigens. Antisera from A. baumannii infected mice demonstrated reactivity to rAMEV2, and subcutaneous immunization of mice with rAMEV2 produced high antibody titer against the construct as well as peptide components. Immunization results in increased frequency of IL-4-secreting splenocytes indicative of a Th2 response. AMEV2-immunized mice were protected against intranasal challenge with a hypervirulent strain of A. baumannii and demonstrated reduced bacterial burden at 48 h. In contrast, all mock vaccinated mice succumbed to infection within 3 days. Results presented here provide insight into the effectiveness of immunoinformatic-based vaccine design and its potential as an effective strategy to combat the rise of MDR pathogens.

show abstract

An improved method for the heterologous production of soluble human ribosomal proteins in Escherichia coli

Cited by 13 publications

References 30 publications

Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids

Characterize direct protein interactions with enrichable, cleavable and latent bioreactive unnatural amino acids

Catalytically self‐sufficient CYP116B5: Domain switch for improved peroxygenase activity

Development and Evaluation of an Immunoinformatics-Based Multi-Peptide Vaccine against Acinetobacter baumannii Infection

Contact Info

Product

Resources

About