Andreas Michael P, Giessen Tobias W
Department of Biomedical Engineering, University of Michigan Medical School, Ann Arbor, MI, USA.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, USA.
MethodsX. 2022 Jul 16;9:101787. doi: 10.1016/j.mex.2022.101787. eCollection 2022.
In recent years a large number of encapsulin nanocompartment-encoding operons have been identified in bacterial and archaeal genomes. Encapsulin-encoding genes and operons from GC-rich Gram-positive bacteria, particularly of the phylum Actinobacteria, are often difficult to overexpress and purify in a soluble form using standard expression systems Here, we present a protocol to heterologously overexpress encapsulin nanocompartments and encapsulin-containing operons in Successful encapsulin production begins with the transfer of a expression plasmid, encoding the gene(s) of interest, via conjugation to the model actinobacterium After growing the conjugated culture to the optimal optical density, protein production is induced by the addition of the inducer thiostrepton, followed by expression in liquid culture for 1-3 days. Cells are lysed and encapsulin proteins purified using ammonium sulfate precipitation and size exclusion chromatography. The method outlined in this protocol can be utilized to improve cargo loading and overall soluble expression of encapsulin systems when compared to expression in •Clone an encapsulin-encoding gene or operon into a expression vector.•Transfer the expression vector to via conjugation.•Heterologously express and purify empty or cargo-loaded encapsulins from
近年来，在细菌和古菌基因组中发现了大量编码封装菌素纳米隔室的操纵子。来自富含GC的革兰氏阳性细菌，特别是放线菌门的编码封装菌素的基因和操纵子，使用标准表达系统通常难以以可溶形式进行过表达和纯化。在此，我们提出了一种在[具体细菌名称]中异源过表达封装菌素纳米隔室和含封装菌素操纵子的方案。成功的封装菌素生产始于通过接合将编码感兴趣基因的表达质粒转移到模式放线菌[具体细菌名称]中。将接合后的培养物生长至最佳光密度后，通过添加诱导剂硫链丝菌素诱导蛋白质生产，随后在液体培养物中表达1 - 3天。细胞裂解后，使用硫酸铵沉淀和尺寸排阻色谱法纯化封装菌素蛋白。与在[其他表达系统]中表达相比，本方案中概述的方法可用于改善封装菌素系统的货物装载和整体可溶性表达。
•将编码封装菌素的基因或操纵子克隆到[具体表达载体]表达载体中。
•通过接合将[具体表达载体]转移到[具体细菌名称]。
•从[具体细菌名称]中异源表达并纯化空载或载有货物的封装菌素。
