His tagged protein purification kit

Manufactured by Beyotime

Sourced in China

The His-tagged protein purification kit is a laboratory equipment designed to facilitate the isolation and purification of proteins containing a histidine-tag (His-tag) sequence. The kit includes all the necessary components to perform efficient protein purification, such as affinity chromatography resins, buffers, and protocols. This product is intended to simplify the process of obtaining pure, tagged proteins for various research applications.

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Lab products found in correlation

Emsa probe biotin labeling kit, by Beyotime (1 mentions)

4 protocols using his tagged protein purification kit

Purification and EMSA of PeSTZ1 Fusion Proteins

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The HIS‐PeSTZ1 fusion proteins were obtained from in vitro prokaryotic expression. The cDNAs encoding full‐length PeSTZ1 were cloned into PET28a to generate His‐fusion recombinant vectors, which were then expressed in Escherichia coli BL21. Then, 0.2 mm isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) was applied to induce the protein. His‐fusion proteins were purified with a His‐Tagged Protein Purification kit (Beyotime, GS008 and GS009, China) according to the manufacturer's instructions.
The PeAPX2 promoter fragment containing CATTAACACTG motifs was synthesized by Sangon (Beijing, China). The EMSA Probe Biotin Labeling kit and a Chemiluminescent EMSA kit (GS008 and GS009; Beyotime Biotechnology, Shanghai, China) were used for the subsequent EMSA assays, which were performed according to the manufacturer's protocols. Briefly, biotin‐labelled probes and fusion proteins were mixed in a binding buffer for 30 min at 22 °C. The unlabelled probes were used for probe competition, and the HIS protein was used as a negative control.

He F., Li H., Wang J., Su Y., Wang H., Feng C., Yang Y., Niu M., Liu C., Yin W, & Xia X. (2019). PeSTZ1, a C2H2‐type zinc finger transcription factor from Populus euphratica, enhances freezing tolerance through modulation of ROS scavenging by directly regulating PeAPX2. Plant Biotechnology Journal, 17(11), 2169-2183.

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Purification of ActA and LDH from S. mutans

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actA and ldh were amplified from S. mutans genomic DNA by PCR and purified. The products were digested by EcoRI and XhoI and then cloned into the expression vector pET28a (Novagen) with an N-terminal fusion of a 6×His tag. Next, the reconstructed plasmids were transformed into E. coli BL21(DE3) cells. The proteins were purified, and their concentrations were determined as described previously (61 (link)). First, overnight cultures of the transformant were diluted 1:20 with fresh LB medium containing 50 μg/mL of kanamycin until an OD_600nm of 0.6 was achieved. After further growth with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at 37°C for 6 h to induce protein expression, the cell pellets were harvested and lysed via sonication. Then, the recombinant ActA and LDH were purified using a His-tagged protein purification kit (Beyotime, Shanghai, China) as per the manufacturer’s instructions and were concentrated via 10 kDa MWKO ultrafiltration (Millipore Amincon, Merck, Germany) from the cell debris. The purified ActA and LDH were confirmed by SDS-PAGE and stored at −80°C for future use.

Ma Q., Pan Y., Chen Y., Yu S., Huang J., Liu Y., Gong T., Zhang Q., Sun Q., Zou J, & Li Y. (2022). Acetylation of Lactate Dehydrogenase Negatively Regulates the Acidogenicity of Streptococcus mutans. mBio, 13(5), e02013-22.

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Cloning and Purification of ActG Protein

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actG was amplified from S. mutans genomic DNA by PCR and purified, digested by NcoI and XhoI, and cloned into the expression vector pET28a (Novagen) with an N-terminal fusion of 6×His-tag. Next, the reconstructed plasmid was cloned to the E. coli BL21 (DE3) cells. Proteins were purified, and concentrations were determined as described previously [55 (link)]. First, overnight cultures of the transformant were diluted 1:20 with fresh LB medium containing 50 μg/mL of kanamycin until OD_600nm of 0.6 was achieved. After further growth with 1-mM isopropyl-β-D-thiogalactopyranoside (IPTG) that induced protein expression, the cell pellets were harvested and lysed by sonification. Then, the recombinant ActG was purified using a His-tagged protein purification kit (Beyotime, Shanghai, China) and concentrated by 5-kDa MWKO ultra-filtration (Millipore Amincon, Merck, Germany) from the cell debris. The purified ActG was confirmed by SDS-PAGE and stored at -80°C for future use.

Ma Q., Pan Y., Chen Y., Yu S., Huang J., Liu Y., Gong T., Zou J, & Li Y. (2021). Acetylation of glucosyltransferases regulates Streptococcus mutans biofilm formation and virulence. PLoS Pathogens, 17(12), e1010134.

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Heterologous Expression and Purification of A6180

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The constructed vector harboring A6180 (pET22b-A6180) was propagated in TOP10. For heterologous expression of recombinant proteins in E. coli, pET22b-A6180 was transformed into E. coli BL21 (DE3) competent cells. Positive colonies were screened using ampicillin and designated as BL21-A6180. To obtain sufficient recombinant protein, the optimal induction conditions were tested at different temperatures (15°C and 37°C) with various doses of isopropyl-β-D-thiogalactopyranoside (IPTG) (1.0 and 0.2 mmol/L). The recombinant protein was purified using a His-tagged protein purification kit (Beyotime, Shanghai, China) for assays of enzymatic properties.

Zou G., Ren J., Wu D., Zhang H., Gong M., Li W., Zhang J, & Yang Y. (2021). Characterization and Heterologous Expression of UDP-Glucose 4-Epimerase From a Hericium erinaceus Mutant with High Polysaccharide Production. Frontiers in Bioengineering and Biotechnology, 9, 796278.

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