Preliminary X-ray crystallographic analysis of Cryptopygus antarticus mannanase and cellulase that were produced through the cold temperature protein expression

Abstract

Biochemical, biophysical, and structural information on proteins provide opportunity for understand their biological functions. One of the limitations to the protein study is the difficulty in obtaining highly purified soluble protein. Protein expression in Escherichia coli (E.coli) at 15-25 ℃ is used to overcome this solubility limitation. However, many recombinant proteins are insolubly expressed even at those low temperatures. In this study, we show that recombinant proteins can be expressed as soluble forms at 6-10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2(DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 L culture grown at 6 and 10 ℃, respectively. These proteins are also suitable for X-ray crystallography. Crystals of CaMan and CaCel diffracted to 2.4 &Aring and 2.6 &Aring resolution, respectively. Consequently, E. coli cultivation at 6&#8211 10 ℃ is an effective method for overcoming a major hurdle of the inclusion body formation.ein expression in Escherichia coli (E.coli) at 15-25 ℃ is used to overcome this solubility limitation. However, many recombinant proteins are insolubly expressed even at those low temperatures. In this study, we show that recombinant proteins can be expressed as soluble forms at 6-10 ℃ without cold adapted chaperon systems. By using E. coli Rosetta-gami2(DE3), we obtained 1.8 and 0.9 mg of Cryptopygus antarticus mannanase (CaMan) and cellulase (CaCel) from 1 L culture grown at 6 and 10 ℃, respectively. These proteins are also suitable for X-ray crystallography. Crystals of CaMan and CaCel diffracted to 2.4 &Aring and 2.6 &Aring resolution, respectively. Consequently, E. coli cultivation at 6&#8211 10 ℃ is an effective method for overcoming a major hurdle of the inclusion body formation.