Backbone circularization of Bacillus subtilis family 11 xylanase increases its thermostability and its resistance against aggregation

Abstract

The activity of proteins is dictated by their three-dimensional structure, the native state, and is influenced by their ability to remain in or return to the folded native state under physiological conditions. Backbone circularization is thought to increase protein stability by decreasing the conformational entropy in the unfolded state. A positive effect of circularization on stability has been shown for several proteins. Here, we report the development of a cloning standard that facilitates implementing the SICLOPPS technology to circularize proteins of interest using split inteins. To exemplify the usage of the cloning standard we constructed two circularization vectors based on the Npu DnaE and gp41-1 split inteins, respectively. We use these vectors to overexpress in Escherichia coli circular forms of the Bacillus subtilis enzyme family 11 xylanase that differ in the identity and number of additional amino acids used for circularization (exteins). We found that the variant circularized with only one additional serine has increased thermostability of 7 °C compared to native xylanase. The variant circularized with six additional amino acids has only a mild increase in thermostability compared to the corresponding exteins-bearing linear xylanase, but is less stable than native xylanase. However, this circular xylanase retains more than 50% of its activity after heat shock at elevated temperatures, while native xylanase and the corresponding exteins-bearing linear xylanase are largely inactivated. We correlate this residual activity to the fewer protein aggregates found in the test tubes of circular xylanase after heat shock, suggesting that circularization protects the protein from aggregation under these conditions. Taken together, these data indicate that backbone circularization has a positive effect on xylanase and can lead to increased thermostability, provided the appropriate exteins are selected. We believe that our cloning standard and circularization vectors will facilitate testing the effects of circularization on other proteins.

Influence of circular permutations on the structure and stability of a six‐fold circular symmetric designer protein

“…This has a dramatic effect on the stability, raising the T m over 10°C and lowering the free energy compared to Pizza6‐AYW by 28.2 kJ/mol. This increased thermal resistance was previously observed with other circularized proteins 22,33 . Unlike the other variants, the CD signal of cPizza6‐AYW does not disappear completely at 218 nm, suggesting this protein retains considerable native structure even after the thermal transition.…”

“…This increased thermal resistance was previously observed with other circularized proteins. 22,33 Unlike the other variants, the CD signal of cPizza6-AYW does not disappear completely at 218 nm, suggesting this protein retains considerable native structure even after the thermal transition. The difference in stability is even greater when considering chemical denaturants.…”

“…cPizza6-AYW was created using a plasmid developed specifically for the gp41-1 split-intein system. 22 All proteins were expressed in E. coli BL21, and the histidine-tagged proteins could be purified with IMAC. Protein purity was verified with SDS-PAGE and analytical size exclusion chromatography (see Figure 2).…”

“…Variants with two (Pizza2) and three (Pizza3) blades were also cloned, in order to test whether the mutants retained the self‐assembling properties of the original Pizza protein. cPizza6‐AYW was created using a plasmid developed specifically for the gp41‐1 split‐intein system 22 …”

“…This vector was created specifically for the purpose of circularizing proteins. 22 All plasmids were transformed into E. coli BL21(DE3) cells, Pizza6-AYW and its variants were expressed and purified following the protocol as described by Noguchi et al 21 Protein expression was induced by adding IPTG to a final concentration of 0.5 mM, and subsequently cells were kept at 20 C for 20 hr. The pellets of proteins containing a hexa-histidine tag were suspended in 50 mM NaH 2 PO4, 200 mM NaCl and 10 mM imidazole.…”