Data on the optimizations of expression and purification of human BiP/GRP78 protein in Escherichia coli

Human BiP/GRP78 is involved in the folding and assembly of proteins in the endoplasmic reticulum. The proteins for crystallization in good amount and quality are prerequisites for obtaining ideal crystals. To meet these requirements, different BiP/GRP78 constructs, competent cells, vectors, and concentrations of inducer were tested in order to obtain soluble BiP/GRP78 protein with the highest amount and best purity. The BiP–T229A–L3,4′–Smt3 fusion protein was expressed in a soluble manner and finally purified with the highest purity using size exclusion chromatography, which was suitable for further protein crystallization.


a b s t r a c t
Human BiP/GRP78 is involved in the folding and assembly of proteins in the endoplasmic reticulum. The proteins for crystallization in good amount and quality are prerequisites for obtaining ideal crystals. To meet these requirements, different BiP/GRP78 constructs, competent cells, vectors, and concentrations of inducer were tested in order to obtain soluble BiP/GRP78 protein with the highest amount and best purity. The BiP-T229A-L 3,4 0 -Smt3 fusion protein was expressed in a soluble manner and finally purified with the highest purity using size exclusion chromatography, which was suitable for further protein crystallization.
& 2016 Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Value of the data
The data indicates that the production of full-length recombinant proteins in the bacterium E. coli can be improved by varying the constructs and concentration of inducer during protein expression. The data indicates that pSMT3 vector may be used to stabilize the full-length recombinant proteins expressed in the bacterium E. coli.
The optimized purification procedure may be still good for the expression of general eukaryotic Hsp70s in the bacterium E. coli.

Data
Fig . 1 shows the SDS-PAGE analysis comparing the induction of the human BiP protein expression tagged with or without Smt3 tag at different conditions. Fig. 2A shows the solubility of the WT, T229A, and T229A-L 3,4 0 BiP-Smt3 fusion proteins. Fig. 2B shows the purifications of BiP proteins (after digestion by Ulp1) on HiTrap Q column, the WT or T229A BiP proteins could not be separated from Smt3, but the T229A-L 3,4 0 -BiP protein could be completely separated from Smt3. Fig. 3 shows the FPLC result and SDS-PAGE analysis for the purified BiP-T229A-L 3,4 0 protein on HiTrap Q column. Fig. 4 shows the crystal picture of BiP-T229A-L 3,4 protein.

The constructs for protein expression
Standard procedure was followed for the construct design according to the description of pET vector cloning strategies [1]. To express BiP protein, the pET-28-BiP construct was designed as below. The BiP (25-630) coding DNA sequence was double digested by NcoI and XhoI restriction enzymes (NEB) in reaction buffer 4, after purified with Gel extraction kit (Qiagen), it was inserted into a predigested (NcoI/XhoI) pET-28(a þ ) vector (Kan R ) carrying the N-terminal His tag by following the manual of In-fusion (HD EcoDry) cloning kit (Clontech, Takara Bio Company).
To express the BiP-Smt3 fusion protein, pSMT3-BiP construct was designed similarly but using pSMT3 vector (a generous gift from Dr. Chris Lima, Sloan-Kettering Institute). In brief, after doubledigesting pSMT3 vector by BamHI and XhoI, BiP (25-630) coding DNA sequence was inserted into pSMT3 vector between these two sites by using the In-fusion (HD EcoDry) cloning kit (Clontech, Takara Bio Company). BamHI recognition sequence (GGATCC) connected Smt3 and BiP coding sequence, which would be translated to Gly and Ser and served as the Ulp1 cleavage site to separate BiP from Smt3 protein.

Protein expression and purification
The competent cell preparation for all different strains including BL21(DE3), BL21(DE3) pLysS, C41 (DE3), and C43(DE3) were essentially the same as described by Agilent Technologies Company and Lucigen Corporation. The pET-28-BiP or pSMT3-BiP DNA was transformed using calcium chloride protocol as previously described [2]. The following day, pick up a single colony and inoculate into 10 ml LB broth containing 25 mg/ml kanamycin or LB broth containing both 25 mg/ml kanamycin and 25 mg/ml chloramphenicol (for BL21(DE3) pLysS transformation). The culture was incubated at 37°C overnight with vigorous shaking, and 1:100 diluted to a fresh medium the next day. When cellular density reached OD 600 ¼ 0.6-0.8, culture would be induced by the addition of IPTG at different concentration (0.25 mM, 0.5 mM, and 1 mM) and incubated at 30°C for another 5-6 h with shaking. 1 ml cell culture was finally collected and resuspended in SDS loading buffer (100 ml per 1 OD 600 ) for SDS-PAGE analysis.
The recombinant BiP-Smt3 proteins (WT, T229A, and T229A-L 3,4 0 ) were purified similarly as mentioned for DnaK-Smt3 protein [3]. First, the fusion proteins passed through a HisTrap column with the buffer containing 2 Â PBS, after removing Smt3 tag by UlP1 protease, the BiP protein was further separated from the cut Smt3 tag on a second HisTrap column. The single phase BiP-T229A-L 3,4 0 protein was acquired by using HiTrap Q column with 25 mM Hepes (pH¼ 7.5) as the buffer. It took several rounds to get the pure BiP-T229A-L 3,4 0 protein in single phase, so the incubation of BiP protein with ATP helped to reach this goal. Finally, the BiP protein was further purified on a Superdex 200 16/60 column and concentrated up to $ 30 mg/ml in buffer A (5 mM Hepes-KOH, pH7.5 and 10 mM KCl). All BiP proteins were flash frozen in the liquid nitrogen before storing at À 80°C freezer.