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Efficient matrix-assisted refolding of the recombinant anti-staphylococcal truncated endolysin LysKCA and its structural and enzymatic description.

Abstract：

:The recombinant truncated endolysin LysK consisting of two catalytic domains, N-terminal CHAP and amidase-2 (LysKCA) 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 LysKCA. 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 LysKCA 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 LysKCA refolding, purification and collection, and also noticeably cuts time and material expenses. The analysis of CD spectroscopy data of LysKCA, 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 LysKCA showing two well-resolved peaks corresponding to the two calorimetrically-revealed domains with the midpoint transition temperature (Tm) 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.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Alaksandr Ž,Sergey G,Maksim P,Sergey K,Niyaz S,Uladzimir P,Mikhail S

doi

10.1016/j.pep.2020.105683

subject

Has Abstract

pub_date

2020-10-01 00:00:00

pages

105683

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(20)30213-8

journal_volume

174

pub_type

杂志文章

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