Efficient soluble expression of secreted matrix metalloproteinase 26 in Brevibacillus choshinensis.
Matrix metalloproteinase 26 (MMP-26) is a novel member of the matrix metalloproteinase family with minimal domain constitution and unknown physiological function. The three-dimensional (3D) structure of the enzyme also remains to be deciphered. Previous studies show that MMP-26 may be expressed in Escherichia coli (E. coli) as inclusion bodies and re-natured with catalytic activity. However, the low re-naturation rate of this method limits its usage in structural studies. In this paper, we tried to clone, express and purify the pro form and catalytic form of MMP-26 (ProMMP-26 and CatMMP-26) in several widely used expression vectors and express the recombinant MMP-26 proteins in E. coli cells. These constructs resulted in insoluble expressions or soluble expressions of MMP-26 with little catalytic activity. We then used Brevibacillus choshinensis (B. choshinensis) as the host system for the soluble and active expression of MMP-26. The enzyme was secreted in soluble form in the supernatant of cell culture medium and purified via a two-step purification process that included Ni(2+) affinity chromatography followed by gel filtration. The yields of purified ProMMP-26 and CatMMP-26 were 12 and 18mg/L, respectively, with high purity and homogeneity. Both ProMMP-26 and CatMMP-26 showed gelatin zymography activity and the purified CatMMP-26 had high enzymatic activity against DQ-gelatin substrate. The large-scale soluble and active protein production for future structural studies of MMP-26 is thus feasible using the B. choshinensis host system.
Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, School of Life Sciences, Jilin University, 2699 Qianjin Street, Changchun, Jilin 130012, PR China.
Tianyang
T
Mu
Weiguo
W
Liang
Ying
Y
Ju
Zhiyong
Z
Wang
Zhongyuan
Z
Wang
Mark D
MD
Roycik
Qing-Xiang Amy
QX
Sang
Dahai
D
Yu
Hongyu
H
Xiang
Xuexun
X
Fang
D000595:Amino Acid Sequence; D001483:Base Sequence; D058675:Brevibacillus / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D053505:Matrix Metalloproteinases, Secreted / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility
D011994:Recombinant Proteins; C410599:MMP26 protein, human; D053505:Matrix Metalloproteinases, Secreted
Brevibacillus choshinensis; Prokaryotic system; Soluble expression