Protein A-mouse acidic mammalian chitinase-V5-His expressed in periplasmic space of Escherichia coli possesses chitinase functions comparable to CHO-expressed protein.
Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.
Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo, Japan.
Akinori
A
Kashimura
Kazuaki
K
Okawa
Kotarou
K
Ishikawa
Yuta
Y
Kida
Kokoro
K
Iwabuchi
Yudai
Y
Matsushima
Masayoshi
M
Sakaguchi
Yasusato
Y
Sugahara
Fumitaka
F
Oyama
D000818:Animals; D016466:CHO Cells; D002688:Chitinases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D051379:Mice; D019897:Periplasm; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; C033223:polyhistidine; D006639:Histidine; C499255:AMCase, mouse; D002688:Chitinases