A simple and efficient expression and purification system using two newly constructed vectors

Abstract

Structural biology places a high demand on proteins both in terms of quality and quantity. Although many protein expression and purification systems have been developed, an efficient and simple system which can be easily adapted is desirable. Here, we report a new system which combines improved expression, solubility screening and purification efficiency. The system is based on two newly constructed vectors, pEHISTEV and pEHISGFPTEV derived from a pET vector. Both vectors generate a construct with an amino-terminal hexahistidine tag (His-tag). In addition, pEHISGFPTEV expresses a protein with an N-terminal His-tagged green fluorescent protein (GFP) fusion to allow rapid quantitation of soluble protein. Both vectors have a tobacco etch virus (TEV) protease cleavage site that allows for production of protein with only two additional N-terminal residues and have the same multiple cloning site which enables parallel cloning. Protein purification is a simple two-stage nickel affinity chromatography based on the His tag removal. A total of seven genes were tested using this system. Expression was optimised using pEHISGFPTEV constructs by monitoring the GFP fluorescence and the soluble target proteins were quantified using spectrophotometric analysis. All the tested proteins were purified with sufficient quantity and quality to attempt structure determination. This system has been proven to be simple and effective for structural biology. The system is easily adapted to include other vectors, tags or fusions and therefore has the potential to be broadly applicable.

Fig. 1. Multiple cloning sites of pEHISTEV /pEHISGFPTEV

Both vectors were constructed containing the f1 replication origin, kanamycin resistance gene, pB332 DNA replication origin, and lac repressor gene. The sequence details of the cloning/expression region show the T7 promoter, lac operator, 6xHis-tag (pEHISTEV) or 6xHis-tagged GFP (pEHISGFPTEV), TEV protease recognition site, restriction edonuclease sites for cloning, and the T7 terminator.

Fig. 2. HisGFP mediated soluble expression screening

A). Colonies of E. coli cells transformed with pEHISGFPTEV constructs (top) expressing GFP-NFIIIhd, RanBP2_2532-2767, Mutase-kp or Sso2226 and with their pEHISTEV constructs (bottom) on the L-agar plate containing 0.4 mM IPTG. We found that the His-tagged and His-GFP colonies could be combined on the same plate and selection was still straightforward. B) Quantitative analysis of soluble expression of the target proteins under the optimised conditions. Measurement of OD₄₈₈ of purified HisGFP is shown on the bottom and the extinction coefficient was calculated based on Beer’s Law. The concentration of the expressed target proteins (after His-GFP removal) was calculated as described in the Materials and Methods section.

Fig. 3. Manual purification of NFIIIhd expressed using pEHISGFPTEV vector

Green fluorescence of purified HisGFPNFIIIhd in elution fractions from first NiAC column (A); SDS-PAGE analysis of the accumulation of HisGFPNFIIIhd (hd) in comparison with the total cellular proteins (B); Elution fraction from the first NiAC column (C) and flow through fractions of second NiAC column after His-GFP removal (D). Numbers on the top of each image indicate the elution fractions. PM: protein molecular weight markers.

Fig. 4. SDS-PAGE analysis of expression and purification of pTP (A), RanBP2_2532-2767 (B) and SP100_181-480 (C) using pEHISTEV vector

pTP, RanBP2_2532-2767 and SP100_181-480 were expressed using pEHISTEV vector in E. coli strain BL21(DE3) and purified as described in the Materials and Methods. Proteins were analysed using a 12% polyacrylamide gel and stained with 0.25% Coomassie blue. The positions of the proteins with His-tag, without His-tag and His-TEV protease are indicated. PM: protein markers, TCP-un, TCP-in: total cellular proteins from un-induced and induced cells, Ni1-pur: protein purified after the first NiAC column, Pro-C: protein after TEV protease cleavage and Ni2-pur: protein purified after the second NiAC.

Fig. 5. SDS-PAGE analysis of expression and purification of Mutase-kp (A), pTP (B) SP100_181-480 (C) RanBP2_2532-2767 (D) and Sso2226 (E) using pEHISGFPTEV vector

Mutase-kp, pTP, SP100_181-480, Sso2226 and RanBP2_2532-2767 were expressed using pEHISGFPTEV vector in E. coli strain BL21(DE3) and purified as described in the Materials and Methods. Proteins were analysed using a 12% polyacrylamide gel and stained with 0.25% Coomassie blue. The positions of the purified proteins and His-GFP are marked by arrows. PM, TCP-un, TCP-in, Ni1-pur, Pro-C and Ni2-pur as for Fig. 4.

Fig. 6. SDS-PAGE analysis of the expression and purification of TYLCV C2 under denaturing conditions

TYLCV C2 gene was expressed using pEHISTEV vector in E. coli strain BL21(DE3). Proteins were analysed using a 15% polyacrylamide gel and stained with 0.25% Coomassie blue. The purified C2 proteins, TEV protease and the cleaved His-tag are indicated. PM: protein markers, TCP: total cellular proteins, SP-f: soluble proteins, Pur-D: purified denatured C2, Pur-re: refolded C2, Pro-C: C2 after TEV protease cleavage and C2-pur: C2 after second NiAC column.

Fig. 7. Automated purification of Sso2226 and Mutase-kp using default setting of protocol AC-DS-GF

A) The full chromatogram for Sso2226 with detection peaks of NiAC1 column (1), DS (2) and GF (3). B) The chromatogram obtained for the final gel filtration step of Sso2226 purification. C) SDS-PAGE analysis of purified Sso2226. Lanes 1-2: samples eluted from NiAC1, lanes 3-5: samples from the superloop and lanes 6-16: samples from FG fractions. D) The chromatogram obtained for the final GF step of Mutase-kp purification. E) SDS-PAGE analysis of purified Mutase-kp. Lanes1-9: samples from GF fractions, lane 10: sample from the superloop and lane 11: total cellular extract. PM: protein markers. F) The chromatogram obtained for the calibration of GF column with protein standards of thyroglobulin (670 kDa), gamma globulin (158 kDa), ovalbumin (44 kDa) and myoglobin (17 kDa).