55750-63-5Relevant articles and documents
Cell-free protein synthesis and: In situ immobilization of deGFP-MatB in polymer microgels for malonate-to-malonyl CoA conversion
Heida, Thomas,Hoefgen, Sandra,K?hler, Tony,Thiele, Julian,Valiante, Vito,Weigel, Niclas
, p. 40588 - 40596 (2020)
In the present work, microgels were utilized as a cell-free reaction environment to produce a functional malonyl-CoA synthetase (deGFP-MatB) under geometry-controlled transcription and translation. Our approach combines the straight-forward optimization of overall protein yield of an E. coli-based cell-free protein synthesis (CFPS) system based on concentration screening of magnesium and potassium glutamate, DNA as well as polyethylene glycol (PEG), and its innovative usage in microgel-based production of a key enzyme of the polyketide synthesis pathway. After partial modification of the carboxyl groups of hyaluronic acid (HA) with 5′-methylfuran groups via 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM)-activation, these were further functionalized with dibenzocyclooctyne (DBCO) and nitrilotriacetic acid (NTA) groups by bio-orthogonal [4+2] Diels-Alder cycloaddition to yield a bifunctional macromer. After coupling the DBCO groups with azide-functionalized DNA, containing the genetic information for deGFP-MatB, via strain-promoted azide-alkyne cycloaddition (SPAAC), the DNA-/NTA-functionalized HA macromer was utilized as base material together with maleimide-functionalized PEG (PEG-mal2) as the crosslinker to form bifunctional microgels utilizing water-in-oil (W/O) microemulsions. As-formed microgels were incubated with nickel sulfate to activate the NTA groups and provide binding sites for deGFP-MatB, which contained six histidine residues (His-tag) for that purpose. The optimized CFPS mixture was loaded into the microgels to initiate the formation of deGFP-MatB, which was detected by a clear increase in fluorescence exclusively inside the microgel volume. Functionality of both, the bound and the decoupled enzyme was proven by reaction with malonate to yield malonyl CoA, as confirmed by a colorimetric assay.
Development of bifunctional anti-PD-L1 antibody MMAE conjugate with cytotoxicity and immunostimulation
Xiao, Dian,Luo, Longlong,Li, Jiaguo,Wang, Zhihong,Liu, Lianqi,Xie, Fei,Feng, Jiannan,Zhou, Xinbo
, (2021/09/27)
In recent years, tumor immunotherapy, especially the combination of PD1/PD-L1 inhibitors and chemotherapy, has developed rapidly. However, the systemic side effects induced by chemotherapy remain a crucial problem that needs to be addressed. Antibody drug conjugates (ADCs) are exceptional target-specific prodrugs that greatly improve the therapeutic window of chemotherapy drugs. Therefore, designing PD-L1-targeting ADCs is an interesting research project. In this study, we confirmed for the first time that the commercial anti-PD-L1 antibody Atezolizumab has better endocytosis efficiencies than Avelumab, and was more suitable for ADC design. Then, the most popular cytotoxic payload MMAE was conjugated to Atezolizumab via a classical dipeptide (valine-alanine) linker to generate a bifunctional PD-L1 ADC (ADC 3). An in vitro cytotoxicity test indicated the potent tumor cell inhibitory activity of ADC 3, with EC50 values of 9.75 nM to 11.94 nM. In addition, a co-culture of PBMCs in vitro proved that ADC 3 retained the immune activation effect of the Atezolizumab antibody. Moreover, ADC 3 exhibited a higher tumor inhibition rate and tumor regression rate in humanized immune system mice. To the best of our knowledge, this is the most active PD-L1-ADC reported thus far, which may promote the development of immunotherapy and novel ADCs.
Method for preparing biotin maleimide
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Paragraph 0025; 0040-0042; 0046; 0048, (2019/04/06)
The invention discloses a method for preparing biotin maleimide. The method comprises the following steps of: 1, reacting 6-maleimidohexanoic acid compound (1) with N-hydroxysuccinimide under the action of a condensing agent to obtain a 6-maleimidohexanoic acid N-hydroxysuccinimide ester compound (2); 2, treating the 6-maleimidohexanoic acid N-hydroxysuccinimide ester compound (2) by using hydrazine hydrate to obtain a 6- maleimidocaproyl hydrazide compound (3); 3, reacting a biotin compound (4) with an acyl halide reagent to obtain a biotinyl acyl halide compound (5); and 4, reacting the 6-maleimidocaproyl hydrazide compound (3) with the biotinyl acyl halide compound (5) to obtain a biotin maleimide compound (6). The method for preparing the biotin maleimide, disclosed by the invention, has the beneficial effects of being suitable for scale-up production, easily available in raw materials, simple in preparation process and friendly to environment.
