High-throughput T7 LIC vector for introducing C-terminal poly-histidine tags with variable lengths without extra sequences

Abstract

Immobilized metal ion affinity chromatography (IMAC) has become one of the most popular protein purification methods for recombinant proteins with a hexa-histidine tag (His-tag) placed at the C- or N-terminus of proteins. Nevertheless, there are always difficult proteins that show weak binding to the metal chelating resin and thus low purity. These difficulties are often overcome by increasing the His-tag to 8 or 10 histidines. Despite their success, there are only few expression vectors available to easily clone and test different His-tag lengths. Therefore, we have modified Escherichia coli T7 expression vector pET21a to accommodate ligation-independent cloning (LIC) that will allow easy and efficient parallel cloning of target genes with different His-tag lengths using a single insert. Unlike most LIC vectors available commercially, our vectors will not translate unwanted extra sequences by engineering the N-terminal linker to anneal before the open reading frame, and the C-terminal linker to anneal as a His-tag.

Figure 1. Cloning Site of pJL and the LIC Reaction Scheme

pJL was derived from pL by changing the MCS by making three different variations to accommodate three different His-tag lengths. These vector variations are made to allow simultaneous cloning of proteins with three different His-tag lengths. The length of the His-tag can be further modified by inserting or removing histidine codons in the PCR primers of the C-terminus LIC linker.

Figure 2. SDS-PAGE Gel of Expression Testing of pJL Clones in BL21(DE3) Star using ZYM autoinducing media

Expression of E. coli serine chemotaxis receptor (Tsr) in pJL vectors. 4–20% SDS-PAGE stained in Coomassie R-250 stain: Lane NC: Negative control, empty pJL-H6 vector; Lane H6: pJL-H6.Tsr; Lane H8: pJL-H8.Tsr; Lane H10: pJL-H10.Tsr. Tsr is a 59 kDa protein. All samples were grown in 2 mL ZYM-5052 media at 37°C, 200 RPM with overnight shaking (~16 hours). Estimated level of expressions are Tsr H6 – 200 mg/L or 25 mg/g of wet cells, Tsr H8 – 100 mg/L or 13 mg/g of wet cells, and Tsr H10 – 200 mg/L or 25 mg/g of wet cells.

Figure 3. IMAC of pJL. Tsr with Different His-tag Lengths

A) UV 280 nm absorbance profile of different His-tag length Tsr elution. H6 – six histidine tag; H8 – eight histidine tag; H10 – ten histidine tag; [Imidazole] – estimated imidazole concentration in mM (corresponds to the right Y-axis scale). The UV absorbance of H10 was scaled up by 1.7X to match integrated peak areas of H6 and H8 for easier comparison of peak height and broadness. As the length of a His-tag increases, the eluting imidazole concentration and broadness of elution peak increases. UV absorbance maxima corresponds to 110 mM (H6), 133 mM (H8), and 180 mM (H10) imidazole. B) 12% SDS-PAGE gel of peak fractions. Each lane corresponds to 3 mL fractions collected at UV absorbance peaks which corresponds to the elution volume, H6 – Lane 1 :12 to 15 mL, Lane 2 : 15 to 18 mL, Lane 3 : 18 to 21 mL; H8 – Lane 1 : 21 to 24 mL, Lane 2 : 24 to 27 mL, Lane 3 : 27 to 30 mL; H10 – Lane 1 : 30 to 33 mL, Lane 2 : 33 to 36 mL, Lane 3 : 36 to 39 mL. Eight micrograms of a total protein, determined by Bradford assay, were loaded on each lane except the last lane (marker lane). The overall purity increases slightly as the length of a His-tag increases indicating a possibility that impurities interact with Tsr.