Miao Tian,Shaowei Huang,Zhiyuan Wang,Junying Fu,Pengmei Lv,Changlin Miao,Tao Liu,Lingmei Yang,Wen Luo
The propeptide is a short sequence that facilitates protein folding. In this study, four highly active Rhizomucor miehei lipase (RML) mutants were obtained through saturation mutagenesis at three propeptide positions: Ser8, Pro35, and Pro47. The enzyme activities of mutants P35 N, P47 G, P47 N, and S8E/P35S/P47A observed at 40 °C, and pH 8.0 were 10.19, 7.53, 6.15, and 8.24 times of that wild-type RML, respectively. The S8E/P35S/P47A mutant showed good thermostability. After incubation at 40 °C for 1 h, 98.98 % of its initial activity remained, whereas wild-type RML retained only 78.76 %. This result indicated that the enhancement of hydrophilicity of 35- and 47- amino-acid residues could promote the interaction between the propeptide and the mature peptide and the enzyme activity and expression level. Highly conserved sites had a more significant impact on enzyme performance than did other sites, similar to the Pro35 and Pro47 mutants showed in this study. This study provides a new idea for protein modification: enzyme performance can be improved through propeptide regulation.
Pulmonary delivery of anti-inflammatory siRNA holds great potential in mitigating the cytokine storm during severe pneumonia. However, commonly utilized polycationic siRNA delivery vehicles can hardly penetrate the mucus barrier, thus greatly hurdling their therapeutic efficacy. Herein, TNF-α siRNA (siTNF-α) delivery nanocomplexes (NCs) are engineered with mucus/cytomembrane dual-penetration capabilities, realized via surface-coating of NCs with RC, an inflammation-sheddable, charge-reversal pro-peptide of RAGE-binding peptide (RBP). RC-coated dendritic poly- L -lysine/siTNF-α (DsT) NCs possess negative surface charges, and can thus efficiently penetrate the mucus layer after intratracheal administration. In the inflamed alveolar space with mild acidity, RC recovers to the cationic RBP and shed off, re-exposing the DsT NCs that efficiently transfect the alveolar macrophages and provokes TNF-α silencing. Thus, siTNF-α and RBP cooperatively alleviate the uncontrolled inflammation during acute lung injury. This study renders a unique approach for mediating trans-mucus nucleic acid delivery, and will find promising utilities for the treatment of severe pneumonia.
Zitao Li,Shuang Xing,Jing Liu,Xiaocong Wu,Sichao Zhang,Di Ma,Xianwei Liu
Microbial transglutaminase (MTG) is a usable enzyme for biomacromolecule modification. In the present study, a “molecular chaperonin” strategy was developed to produce MTG in E. coli cytoplasm with high expression level and a “small molecule-mediated chemical modification” strategy was adopted to strip propeptide chaperonin efficiently during purification . Propeptide (Pro) was expressed separately as a chaperonin to facilitate MTG expression in E. coli cytoplasm with a yield up to 300 mg or about 9 kU from 1 L fed-batch culture. Furthermore, small molecular chemicals were applied to interfere the interaction between MTG and Pro. Chemical acetylation was identified as a suitable method to strip Pro resulting in pure MTG with high specific activity up to 49.6 U/mg. The purified acetylated MTG was characterized by MS analysis. The deconvoluted mass and Peptide Sequence Tags analysis confirmed acetylation on amino groups of MTG protein. Finally, the applications of obtained MTG were demonstrated via protein polymerization of bovine serum albumin and PEGylation of human interferon-α2b. Our method provides MTG with high purity and specific activity as well as unique merit with masked amino groups thus avoiding self-polymerization and cross-linking between MTG and substrates.
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