The recombinant truncated endolysin LysK consisting of two catalytic domains, N-terminal CHAP and amidase-2 (LysK_CA) was overexpressed in E. coli in the form of inclusion bodies (IBs). These IBs were dissolved in 6 M solution of urea followed by the refolding process. The refolding efficacy of the dilution and matrix-assisted renaturation method on SP Sepharose was compared at different purification stages of LysK_CA. Solubilizate of IBs, DEAE Sepharose flowthrough, and SP Sepharose elution fractions were examined. The presence of negatively charged nucleic acids (NA) in the solution has shown a decrease in the recombinant LysK_CA refolding yield (less than 11.5 ± 1.3% for both renaturation methods) due to their non-specific interaction with the positively charged endolysin. The renaturation efficiency of the enzyme purified from NA (SP elution fraction) was about 29.5 ± 6.7% and 28.2 ± 3.75% for dilution and matrix-assisted methods respectively. The later approach allows conducting one-step LysK_CA refolding, purification and collection, and also noticeably cuts time and material expenses.

The analysis of CD spectroscopy data of LysK_CA, renatured on the resin matrix, revealed alpha helices and beta strands content similar to that of the modeled 3D structure. The theoretical 3D model with two predicted domains (CHAP and amidase-2) agrees well with the differential scanning calorimetry (DSC) results of the renatured LysK_CA showing two well-resolved peaks corresponding to the two calorimetrically-revealed domains with the midpoint transition temperature (T_m) of 40.1 and 65.3°С. The enzyme so obtained exhibited in vitro anti-staphylococcal activity with 2.3 ± 0.45 × 10³ U/mg and retained it for at least one year.

由两个催化结构域，N端CHAP和酰胺酶2（LysK _CA）组成的重组截短的溶素LysK以包涵体（IBs）的形式在大肠杆菌中过表达。将这些IB溶于6 M尿素溶液中，然后进行复折过程。比较了在不同的LysK _CA纯化阶段，稀释液和基质辅助复性法在SP Sepharose上的重折叠效果。检查了IBs，DEAE Sepharose流通液和SP Sepharose洗脱级分的溶解度。溶液中带负电荷的核酸（NA）的存在已显示重组LysK _CA的减少由于它们与带正电的细胞内溶素的非特异性相互作用，导致复性收率（两种复性方法均低于11.5±1.3％）。从NA纯化的酶（SP洗脱级分）的复性效率对于稀释法和基质辅助法分别约为29.5±6.7％和28.2±3.75％。后面的方法允许进行LysK _CA的一步重折叠，纯化和收集，还可以显着减少时间和材料费用。

在树脂基质上变性的LysK _CA的CD光谱数据分析表明，α螺旋和β链含量与建模的3D结构相似。具有两个预测域（CHAP和酰胺酶2）的理论3D模型与变性LysK _CA的差示扫描量热法（DSC）结果非常吻合，显示出两个很好分辨的峰，对应于两个具有中点转变温度的量热揭示域（T _m）为40.1和65.3°С。如此获得的酶具有2.3±0.45×10 ³ U / mg的体外抗葡萄球菌活性，并保留了至少一年。