Bacillus subtilis has become an increasingly popular host for recombinant protein expression. With its ability to secrete protein directly into culture media, amenability to medium- and large-scale fermentation, lack of codon bias, and designation by the U.S. Food and Drug Administration as an organism that is Generally Regarded As Safe (GRAS), it's no wonder that the majority of industrially-produced enzymes are expressed in Bacillus species such as B. subtilis. Optimization of secretion, however, can be necessary to achieve highest yields. To address this, the B. subtilis Secretory Protein Expression System from Takara Bio allows rapid development of a library of B. subtilis clones, each bearing a pBE-S construct in which the ORF for your protein of interest is fused with sequences for 173 unique signal peptides. Perform a downstream assay to identify and select clones which secrete the highest amount of functional protein into the culture media, and you can quickly identify which signal peptide results in the most efficient expression of your desired secreted protein.

NOTE: For commercial use of this product, please fill out the commercial use inquiry form. Upon submission of the form, you will be directed to a page with appropriate ordering information.

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Flowchart of the experimental procedure for the B

Flowchart of the experimental procedure for the B. subtilis Secretory Protein Expression System.

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3380: B. Subtilis Secretory Protein Expression System

The Human Cell-Free Protein Expression System from Takara Bio is easy to use. The single-tube reaction is easily assembled and protein synthesis is complete in as little as 1 hr at 32°C. The Human Cell-Free Protein Expression System provides high yield (e.g., 50 μg/ml of human eIF4G) of functional protein, including proteins requiring modifications such as glycosylation, phosphorylation, or fatty acylation and even with large proteins (over 150 kDa). Genes of interest may be cloned rapidly into pT7-IRES vectors using In-Fusion cloning technology. After expression, proteins with N-terminal or C-terminal His tag or N-terminal Myc tag can be generated, depending on choice of vector. 

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Principle of the Human Cell-Free Protein Expression System

Principle of the Human Cell-Free Protein Expression System. A) In an easy and simple protocol, target protein translation is initiated by adding pT7-IRES Vector containing the target gene cassette and other kit components. B) The target gene RNA transcribed from the pT7-IRES Vector has an IRES sequence designed to promote protein translation initiation. As protein synthesis progresses, the translation initiation factor from the cell lysate becomes inactivated. The translation enhancement factor in the reaction mixture, however, reactivates this inactivated translation initiation factor and thereby maintains high translation level.

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Time course of beta-galactosidase expression and effect of translation enhancement factor

Time course of beta-galactosidase expression and effect of translation enhancement factor. Eight replicates of beta-galactosidase in vitro translation were performed using 1 µl of Control Vector per reaction. At each of the indicated time points (0, 0.5, 1, 2, 3, 4.5, 6, and 8 hours after start of the reaction), one reaction tube was removed and used for beta-galactosidase activity assay with O-nitrophenyl-b-D-galactopyranoside (ONPG) as the substrate. A separate set of reactions were conducted in absence of the translation enhancement factor (Mixture-2). The activity of beta-galactosidase increased over time to peak at approximately 4.5 h. Additionally, the presence of Mixture-2 containing translation enhancement factor markedly increased yield of active protein.

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Synthesis of high molecular weight proteins using the Human Cell-Free Protein Expression System

Synthesis of high molecular weight proteins using the Human Cell-Free Protein Expression System. In vitro translation reactions were performed to synthesize human Dicer (200 kDa, lane 2) or human eIF4G (170 kDa, lane 3) protein. Reactions were analyzed by SDS-PAGE and coomassie blue staining. Arrowheads indicate target proteins. Lane 1, negative control.

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3281: Human Cell-Free Protein Expression System

Capturem Protein A Buffer is used for purification of IgGs on Capturem Protein A Miniprep Columns (Catalog # 635717), Capturem Protein A 96-Well Plate (Catalog # 635716), Capturem Protein A 24-Well Plate - HC (Catalog # 635743), and Capturem Protein A Maxiprep Columns (Catalog # 635720).

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635746: Capturem Protein A Buffer

Takara CBB Protein Safe Stain is a highly sensitive protein staining reagent based on Coomassie Brilliant Blue G-250. This product can be used to stain SDS-PAGE or native PAGE gels quickly. It allows the visualization of protein bands approximately 5 minutes after the staining process begins, and provides maximum sensitivity after 60 minutes of staining. Stained gels may be destained with deionized water to remove background staining. Takara CBB Protein Safe Stain can detect bands containing as little as 8 ng of protein. It can be handled safely, since it does not contain methanol or acetic acid.

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Using TaKaRa CBB Protein Safe Stain to stain a 12% SDS-PAGE gel

Using TaKaRa CBB Protein Safe Stain to stain a 12% SDS-PAGE gel.

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T9320A: TaKaRa CBB Protein Safe Stain

CLEARLY Stained Protein Ladder is a mixture of 11 pre-stained recombinant highly purified proteins. When this ladder is analyzed by SDS-PAGE, it is resolved into clearly identifiable visible bands from 10 to 210 kDa. The 30 and 70 kDa bands have quadruple the mass of the other bands and serve as reference indicators. This ladder is pre-mixed with loading buffer and ready to load. Pre-heating is not required. 10 μl of this ladder is loaded per lane of SDS-PAGE minigel for standard use. In this case, this ladder is sufficient for 50 lanes. This ladder is useful for monitoring of protein migration during SDS-PAGE and verifying transfer efficiency at western blotting.^＊

＊: Because the covalent coupling of the dye to the proteins affects their electrophoretic behavior on SDS-PAGE gels relative to unstained proteins, this ladder is not recommended for use as a precise size standard when performing SDS-PAGE to calculate the molecular weight of a protein of interest.

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Migration pattern of CLEARLY Stained Protein Ladder (Cat.# 3454A) on an 12% SDS-PAGE gel

Migration pattern of CLEARLY Stained Protein Ladder (Cat.# 3454A) on an 12% SDS-PAGE gel.

CLEARLY Stained Protein Ladder is a mixture of 11 pre-stained recombinant highly purified proteins. When this ladder is analyzed by SDS-PAGE, it is resolved into clearly identifiable visible bands from 10 to 210 kDa. The 30 and 70 kDa bands have quadruple the mass of the other bands and serve as reference indicators. This ladder is pre-mixed with loading buffer and ready to load. Pre-heating is not required. 10 μl of this ladder is loaded per lane of SDS-PAGE minigel for standard use. In this case, this ladder is sufficient for 50 lanes. This ladder is useful for monitoring of protein migration during SDS-PAGE and verifying transfer efficiency at western blotting.^＊

＊: Because the covalent coupling of the dye to the proteins affects their electrophoretic behavior on SDS-PAGE gels relative to unstained proteins, this ladder is not recommended for use as a precise size standard when performing SDS-PAGE to calculate the molecular weight of a protein of interest. Cat. # 3454B contains 3 of Cat. # 3454A. Please refer to Cat. # 3454A for complete product documentation and resources.

Selecting a blocking buffer that best suppresses non-specific signals is critical to obtaining clear, specific Western blot results. This product is completely chemically defined and free of protein components. Because of this, the Western BLoT Blocking Buffer (Protein Free) provides higher sensitivity than other commonly used blocking agents, such as nonfat milk. In addition to detection of normal proteins, this blocking buffer is excellent for the detection of phosphorylated proteins. In addition, completely protein-free assays are possible when this product is used in combination with Western BLoT Immuno Booster PF (Cat. #T7115A). To obtain the highest detection sensitivity, combine with the Western BLoT HRP Substrate series.

CLEARLY Protein Ladder (Unstained) is a mixture of 11 recombinant highly purified proteins. When this ladder is analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250, it is resolved into quite clearly identifiable bands from 10 to 250 kDa. This ladder is intended for use as a precise size standard when performing SDS-PAGE to calculate the molecular weight of a protein of interest. The 30 and 80 kDa bands have quadruple the mass of the other bands and serve as reference indicators. This ladder is pre-mixed with loading buffer and ready to load. Preheating is not required. 5 μl of this ladder is loaded per lane of SDS-PAGE minigel for standard use. In this case, this ladder is sufficient for 100 lanes.

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Migration pattern of CLEARLY Protein Ladder (Unstained) (Cat.# 3453A) on a 12% SDS-PAGE gel.

Migration pattern of CLEARLY Protein Ladder (Unstained) (Cat.# 3453A) on a 12% SDS-PAGE gel.

CLEARLY Protein Ladder (Unstained) is a mixture of 11 recombinant highly purified proteins. When this ladder is analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250, it is resolved into quite clearly identifiable bands from 10 to 250 kDa. This ladder is intended for use as a precise size standard when performing SDS-PAGE to calculate the molecular weight of a protein of interest. The 30 and 80 kDa bands have quadruple the mass of the other bands and serve as reference indicators. This ladder is pre-mixed with loading buffer and ready to load. Pre-heating is not required. 5 μl of this ladder is loaded per lane of SDS-PAGE minigel for standard use. In this case, this ladder is sufficient for 100 lanes. Cat. # 3453B contains 3 of Cat. # 3453A. Please refer to Cat. # 3453A for complete product documentation and resources.

Takara's Protein Molecular Weight Markers are designed for use in SDS Polyacrylamide gel electrophoresis. These markers come in three ranges: Low (Molecular weight range: 14.3–97.2 kDa), High (Molecular weight range: 44.3–200 kDa) and Broad (Molecular weight range: 6.5–200 kDa). Each protein in these markers is quantitated to yield uniform band intensities when stained with Coomassie Brilliant Blue R-250 after running on an SDS polyacrylamide gel. Five μl of 20-fold diluted marker is recommended per lane of SDS-PAGE minigel for standard use.

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3451: Protein Molecular Weight Marker (High)