Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

Ahsan, Fatima; Gardner, Qurratulann Afza; Rashid, Naeem; Towers, Greg J.; Akhtar, Muhammad

doi:10.1016/j.bioorg.2017.11.016

Cited by 5 publications

(2 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this context, we also analyzed their dependence on their most direct regulator (glucose) and some direct effectors (deacetylase CobB, peptidyl N(ε)-Lysine acetyl-transferase PatZ). Although this topic belongs to the ambit of molecular physiology, the clarification of these mechanisms could have important consequences in the field of biotechnological applications (Kuczynska-Wiśnik et al, 2016 ; Ahsan et al, 2017 ; Basak et al, 2017 ; Ishigaki et al, 2017 ; Venkat et al, 2018 ), and even for some medical issues (Bernal et al, 2016 ; Ou et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Guebel¹,

Torres

2018

Front. Microbiol.

View full text Add to dashboard Cite

Background: While in eukaryotes acetylation/deacetylation regulation exerts multiple pleiotropic effects, in Escherichia coli it seems to be more limited and less known. Hence, we aimed to progress in the characterization of this regulation by dealing with three convergent aspects: the effector enzymes involved, the master regulator CRP, and the dependence on glucose availability.Methods: The transcriptional response of E. coli BW25113 was analyzed across 14 relevant scenarios. These conditions arise when the wild type and four isogenic mutants (defective in deacetylase CobB, defective in N(ε)-lysine acetyl transferase PatZ, Q- and R-type mutants of protein CRP) are studied under three levels of glucose availability (glucose-limited chemostat and glucose-excess or glucose-exhausted in batch culture). The Q-type emulates a permanent stage of CRPacetylated, whereas the R-type emulates a permanent stage of CRPdeacetylated. The data were analyzed by an optimized factorial microarray method (Q-GDEMAR).Results: (a) By analyzing one mutant against the other, we were able to unravel the true genes that participate in the interaction between ΔcobB/ΔpatZ mutations and glucose availability; (b) Increasing stages of glucose limitation appear to be associated with the up-regulation of specific sets of target genes rather than with the loss of genes present when glucose is in excess; (c) Both CRPdeacetylated and CRPacetylated produce extensive changes in specific subsets of genes, but their number and identity depend on the glucose availability; (d) In other sub-sets of genes, the transcriptional effect of CRP seems to be independent of its acetylation or deacetylation; (e) Some specific ontology functions can be associated with each of the different sets of genes detected herein.Conclusions: CRP cannot be thought of only as an effector of catabolite repression, because it acts along all the glucose conditions tested (excess, limited, and exhausted), exerting both positive and negative effects through different sets of genes. Acetylation of CRP does not seem to be a binary form of regulation, as there is not a univocal relationship between its activation/inhibitory effect and its acetylation/deacetylation stage. All the combinatorial possibilities are observed. During the exponential growth phase, CRP also exerts a very significant transcriptional effect, mainly on flagellar assembly and chemotaxis (FDR = 7.2 × 10−44).

show abstract

Section: Discussionmentioning

confidence: 99%

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Guebel¹,

Torres

2018

Front. Microbiol.

View full text Add to dashboard Cite

show abstract

“…It was reported that acetylation could occur at N-terminal Cys residue in IFNα2b, and the acetylated form had only 10% of the activity of native molecule [17]. To overcome this N-terminal heterogeneity, many strategies have been developed, such as engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal Met [20], and co-expression of engineered MetAP in E. coli [14].…”

Section: Discussionmentioning

confidence: 99%

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

Tao

Zhao

et al. 2020

Molecules

View full text Add to dashboard Cite

Recombinant human IFNα2b (rhIFNα2b), as an important immune-related protein, has been widely used in clinic for decades. It is also at the forefront of the recent emergence of biosimilar medicines, with numerous products now available worldwide. Although with the same amino acid sequence, recombinant proteins are generally heterogeneous due to post-translational modification and chemical reactions during expression, purification, and long-term storage, which could have significant impact on the final product quality. So therapeutic rhIFNα2b must be closely monitored to ensure consistency, safety, and efficacy. In this study, we compared seven rhIFNα2b preparations from six manufacturers in China and one in America, as well as four batches of rhIFNα2b preparations from the same manufacturer, measuring IFNα2b variants and site-specific modifications using a developed LC/Q-TOF approach. Three main forms of N-terminus, cysteine, methionine, and acetylated cysteine were detected in five rhIFNα2b preparations produced in E. coli (1E~5E) and one in Pseudomonas (6P), but only the native form with N-terminal cysteine was found in rhIFNα2b preparation produced in Saccharomyces cerevisiae (7Y). Two samples with the lowest purity (4E and 6P), showed the highest level of acetylation at N-terminal cysteine and oxidation at methionine. The level of oxidation and deamidation varied not only between samples from different manufacturers but also between different batches of the same manufacturer. Although variable between samples from different manufacturers, the constitution of N-terminus and disulfide bonds was relatively stable between different batches, which may be a potential indicator for batch consistency. These findings provide a valid reference for the stability evaluation of the production process and final products.

show abstract

Molecular and Biological Characterization of a Prepared Recombinant Human Interferon Alpha 2b Isoform

Shaaban

El‐Sayed

Samir

et al. 2018

Appl Biochem Biotechnol

View full text Add to dashboard Cite

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

Cited by 5 publications

References 20 publications

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Influence of Glucose Availability and CRP Acetylation on the Genome-Wide Transcriptional Response of Escherichia coli: Assessment by an Optimized Factorial Microarray Analysis

Analysis of Molecular Heterogeneity in Therapeutic IFNα2b from Different Manufacturers by LC/Q-TOF

Molecular and Biological Characterization of a Prepared Recombinant Human Interferon Alpha 2b Isoform

Contact Info

Product

Resources

About