An efficient procedure for purification of recombinant human β heat shock protein 90

Abstract

Background and the purpose of the study: Heat Shock Protein 90 (Hsp90) is typically the most abundant chaperone in the eukaryotic cell cytoplasm, and its expression is essential for loading immunogenic peptides onto major histocompatibility complex molecules for presentation to T-cells. Therefore, it may act as a good candidate as an adjuvant molecule in vaccine technology.

Methods: Initially the human Hsp90β gene was cloned into the heat inducible expression vector pGP1-2 and then the recombinant protein was isolated by ion exchange chromatography. After intradermal injection of confirmed purified band of protein to rabbits and isolation of the serum IgG antibody, for its affinity purification, the rabbit's purified Hsp90 specific IgG was coupled to the cyanogen bromide-activated Sepharose 4B.

Results: The recovery of the purified protein of interest by affinity chromatography was 50%.

Conclusion: This research enabled purification of human heat shock protein by a laboratory prepared column chromatography.

Keywords: CNBr activated Sepharose 4B; Head shock protein 90(Hsp90); Rabbit IgG; pGP1-2.

Figure 1

PCR product of Hsp90 on 1% agarose gel Lane 1: 100 bp DNA Ladder (Fermentas, Germany) Lane 2: 2174 bp Hsp90 PCR product

Figure 2

pGp1-2 subcloning confirmation Lane 1: Undigested cloned plasmid Lane 2: Gene Ruler 1kb DNA Ladder (Fermentas, Germany) Lane 3: Digested plasmid by DraII (linear form)

Figure 3

Confirmation of Hsp90 Cloning into the pGP1-2 by specific PCR Lane a: Gene Ruler 1kb DNA Ladder (Fermentas, Germany) Lane b: Direct PCR product of cloned plasmid

Figure 4

Showing Hsp90β subunit purification chromatogram of fractions obtained from ion exchange chromatography of expressed construct a, b: Loading & washing stages c: Elution stage d, e, f: Washing stage

Figure 5

10% SDS-PAGE of obtained fractions which are the peaks signed in Fig4.It was showed 90kDa protein band in lane c.

Figure 6

Chromatogram of IgG purification from serum by protein A affinity chromatography 1- 3 –11: Loading 2- 12- 22: Washing 3- 23 – 25: Elution 4- 26–30: Washing

Figure 7

10% SDS-PAGE of Ig G after Protein A affinity chromatography Lane a: Total serum proteins Lane b: Boiling with SDS sample buffer Lane c: Purified IgG

Figure 8

Immuno diffusion test using injected rabbit serum (+) and negative control (−) 1- Rabbit's serum without injection 2- Purified Hsp90 band by gel extraction 3- Immunized rabbit's serum

Figure 9

Western Blotting of recombinant protein purified by βHsp90- specific sepharose 4B using polyclonal antibody