Escherichia coli bl21 cells

Cloning, expression and purification of recombinant SjTPx-1 (rSjTPx-1) were carried out as previously described with some modifications [4 (link)]. Briefly, first strand synthesis of
complementary DNA (cDNA) was done using PrimeScript^TM(Takara Bio. Inc., Otsu). Forward primer 5′-GC GGA TCC ATG GTA CTG ATT CCA AAT-3′ and reverse primer: 5′-GC CTC
GAG TAA TCA GTG ATT CAC TTT-3′ with BamHI/XhoI as the restriction sites (underlined) were used for polymerase chain reaction (PCR) amplification of the double strand DNA.
The DNA fragment was cloned into pET-28a (+) vector (Novagen, Madison, WI, U.S.A.) for expression of the recombinant protein with 6xHis-tag on the N terminal in Escherichia coli (BL21) cells (Takara Bio
Inc.). rSjPTx-1 was purified with affinity chromatography nickel nitrilotriacetic acid (Ni-NTA) protein purification kit (Qiagen, Hilden, Germany) and dialyzed against cold phosphate buffered saline (PBS) (pH 7.4) prior
to use. The recombinant protein was quantified by Pierce^TM BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, U.S.A.). Purity of rSjTPx-1 was assessed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE).

In order to prepare human recombinant sFcγRIIB, PET-sFcγRIIB plasmid from our laboratory was transformed into competent Escherichia coli BL21 cells (Takara Bio, Inc.). To prepare mouse recombinant sFcγRIIB, mouse sFcγRIIB gene was cloned using TRE-sFcγRIIB plasmid as a template and sFcγRIIB primers (forward primer, 5′-GGA ATT CAT GGG AAT CCT GCC GTT CCT ACT GA-3′ and reverse primer, 5′-CCC AAG CTT TGT CAA TAC TGG TAA AGA CCT GCT G-3′), and was inserted into pET-32a (+) (Beijing Solarbio Science & Technology Co., Ltd.) vector to construct the PET-sFcγRIIB plasmid. The PET-sFcγRIIB plasmid (2 µl) was subsequently was transformed into competent Escherichia coli BL21 cells. Both sets of transformed BL21 cells were induced by IPTG to produce sFcγRIIB proteins, which were purified using a His Bind^® Purification kit (Merck & Co., Inc.) according to the manufacturer's instructions. Purified sFcγRIIB proteins were validated by western blot analysis.