Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae

Figures & data

Figure 1. PCR amplification and restriction digestion of S. cerevisiae rpb5 gene. Lane 1: DNA ladder. Lane 2: PCR product (648 bp). Lane 3: pSK+-rpb5 digested with Bam HI & Hind III; upper band (3 Kb) is pSK+ vector and lower band (648 bp) is the rpb5 gene. Lane 4: pET28a(+)-rpb5 was restricted with BamHI & Hind III; upper band (5.3 Kb) shows pET28a vector while the lower band (648 bp) shows rpb5 gene. 1 % agarose gel electrophoresis was used to visualize the bands.

Figure 2. Rpb5 protein expression, solubility and purification analysis. Protein samples were separated by 12 % SDS-PAGE, and stained with CBB. Lane 1: Molecular weight marker. Lane 2: Rpb5 Un-induced control lysate. Lane 3: Rpb5 Induced lysate. Lane 4: Rpb5 protein pellet fraction after lysis. Lane 5: Soluble fraction after lysis. Lane 6: purified Rpb5 protein.

Table 1. Amount of cells and purification fold after IMAC.

Crude cell pellet/L culture (g)	Total protein/L culture (mg)	Protein yield after IMAC/L culture (mg)
∼2.83	∼240.8	∼45

Figure 3. Western blot analysis on PVDF membrane. Lane 1, Molecular weight marker. Lane 2, Rpb5 recombinant protein detected by western blotting with a mouse anti-His antibody.