The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E. coli

Fin, Nafiseh Alsadat Seyed Hosseini; Barshan-Tashnizi, Mohammad; Sajjadi, Seyed Mehdi; Asgari, Saeme; Mohajerani, Nazanin; Mirzahoseini, Hasan

doi:10.1016/j.pep.2019.01.011

Cited by 5 publications

(3 citation statements)

References 48 publications

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“…In a study by Fin et al, three sets of chaperone plasmids, GroEL-GroES (GroELS), Dnak-Dnaj-GrpE (DnaKJE) and trigger factor (TF), were co-expressed in E. coli BL21 (DE3) in pairs with two pET22-b vectors carrying recombinant hirudin-PA (Hir) and various alkaline phosphatase (PhoA) and L-ASNase II signal sequences. DnaKJE was found to be the only factor that significantly increased L-ASNase expression in the periplasm (p = 0.04) [128].…”

Section: Co-expression With Chaperonesmentioning

confidence: 95%

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Shishparenok,

Gladilina,

Zhdanov

2023

IJMS

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Genetic engineering for heterologous expression has advanced in recent years. Model systems such as Escherichia coli, Bacillus subtilis and Pichia pastoris are often used as host microorganisms for the enzymatic production of L-asparaginase, an enzyme widely used in the clinic for the treatment of leukemia and in bakeries for the reduction of acrylamide. Newly developed recombinant L-asparaginase (L-ASNase) may have a low affinity for asparagine, reduced catalytic activity, low stability, and increased glutaminase activity or immunogenicity. Some successful commercial preparations of L-ASNase are now available. Therefore, obtaining novel L-ASNases with improved properties suitable for food or clinical applications remains a challenge. The combination of rational design and/or directed evolution and heterologous expression has been used to create enzymes with desired characteristics. Computer design, combined with other methods, could make it possible to generate mutant libraries of novel L-ASNases without costly and time-consuming efforts. In this review, we summarize the strategies and approaches for obtaining and developing L-ASNase with improved properties.

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Section: Co-expression With Chaperonesmentioning

confidence: 95%

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Shishparenok,

Gladilina,

Zhdanov

2023

IJMS

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“…The expression enhancement of the smTG in Corynebacterium glutamicum was also observed after fusing with a chimeric pro-region from Streptomyces cinnamoneus transglutaminase [15]. In E. coli, co-expressing with chaperones can benefit enzyme folding process and soluble form [16,17]. As mRNA contain stable elements that can regulate the translation rate and protein folding, and mRNA folding energy may thus be a key factor for protein expression [18].…”

Section: Introductionmentioning

confidence: 92%

Active secretion of a thermostable transglutaminase variant in Escherichia coli

Wang

Zhao

et al. 2022

Microb Cell Fact

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Background Streptomyces mobaraenesis transglutaminase (smTG) is widely used to generate protein crosslinking or attachment of small molecules. However, the low thermostability is a main obstacle for smTG application. In addition, it is still hard to achieve the secretory expression of active smTG in E. coli, which benefits the enzyme evolution. In this study, a combined strategy was conducted to improve the thermostability and secretory expression of active smTG in E. coli. Results First, the thermostable S. mobaraenesis transglutaminase variant S2P-S23V-Y24N-S199A-K294L (TGm1) was intracellularly expressed in pro-enzyme form in E. coli. Fusing the pro-region of Streptomyces hygroscopicus transglutaminase (proH) and TrxA achieved a 9.78 U/mL of intracellular smTG activity, 1.37-fold higher than the TGm1 fused with its native pro-region. After in vitro activation by dispase, the TGm1 with proH yielded FRAPD-TGm1, exhibiting 0.95 ℃ and 94.25% increases in melting temperature and half-life at 60 ℃ compared to FRAP-TGm1 derived from the expression using its native pro-region, respectively. Second, the TGm1 with proH was co-expressed with transglutaminase activating protease and chaperones (DnaK, DnaJ, and GrpE) in E. coli, achieving 9.51 U/mL of intracellular FRAPD-TGm1 without in vitro activation. Third, the pelB signal peptide was used to mediate the secretory expression of active TGm in E. coli, yielding 0.54 U/mL of the extracellular FRAPD-TGm1. A script was developed to shuffle the codon of pelB and calculate the corresponding mRNA folding energy. A 1.8-fold increase in the extracellular expression of FRAPD-TGm1 was achieved by the Top-9 pelB sequence derived from the coding sequences with the lowest mRNA folding energy. Last, deleting the gene of Braun’s lipoprotein further increased the extracellular yield of FRAPD-TGm1 by 31.2%, reached 1.99 U/mL. Conclusions The stabilized FRAPD-smTG here could benefit the enzyme application in food and non-food sectors, while the E. coli system that enables secretory expression of active smTG will facilitate the directed evolution for further improved catalytic properties. The combined strategy (N-terminal modification, co-expression with chaperones, mRNA folding energy optimization of signal peptide, and lipoprotein deletion) may also improve the secretory expression of other functional proteins in E. coli.

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“…Overexpression of chaperone proteins containing HSP60 and HSP70 with recombinant hirudin-PA increased the secretory production by 2.6-fold and 3.5-old in E. coli, respectively (Seyed Hosseini Fin et al, 2019). Co-expression of protein disulfide isomerase improved the secretory production of disulfide-rich proteins (Inan et al, 2006), whereas the secretory production of human parathyroid hormone without disulfide bonds was also increased 10-fold in Pichia pastoris (Vad et al, 2005).…”

Section: Introductionmentioning

confidence: 97%

Enhanced production of recombinant proteins in <i>Corynebacterium glutamicum </i>using a molecular chaperone

Wang

Liu

et al. 2023

J. Gen. Appl. Microbiol.

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Protein synthesis in Corynebacterium glutamicum is critical for applications in biotechnology and medicine. However, the use of C. glutamicum for protein production is limited by its low expression and aggregation. To overcome these limitations, a molecular chaperone plasmid system was developed in this study to improve the efficiency of recombinant protein synthesis in C. glutamicum.The effect of molecular chaperones on target protein synthesis (Single-chain variable fragment, Scfv) under three different promoter strengths was tested. In addition, the plasmid containing the molecular chaperone and target protein was verified for growth stability and plasmid stability. This expression model was further validated using two recombinant proteins, human interferon-beta (Hifn) and hirudin variant III (Rhv3). Finally, the Rhv3 protein was purified, and analysis of Rhv3 activity confirmed that the use of a molecular chaperone led to an improvement in test protein synthesis.Thus, the use of molecular chaperones is believed to will improve recombinant proteins synthesis in C. glutamicum.

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The effects of overexpression of cytoplasmic chaperones on secretory production of hirudin-PA in E. coli

Cited by 5 publications

References 48 publications

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Engineering and Expression Strategies for Optimization of L-Asparaginase Development and Production

Active secretion of a thermostable transglutaminase variant in Escherichia coli

Enhanced production of recombinant proteins in <i>Corynebacterium glutamicum </i>using a molecular chaperone

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