Expression, Purification And Adjuvant Activity Of Human Heat Shock Protein 70D

Heat shock proteins (HSP) are conserved proteins found in all prokaryotes and eukaryotes and highly expressed in the environments of stress stimuli including heat shock, UV radiation, viral or bacterial infection. Being a large family of stress proteins, HSPs are known to play essential roles in protein synthesis and folding as molecular chaperones, It was also reported that some members of HSP family such as HSP70 participate in important immune reaction including antigen presentation and T cell activation. HSP preparations derived from tumor or virus-infected cells are capable of eliciting antigen-specific cellular immunity. HSPs are now regarded as a kind of important molecule in mediating tumor or viral specific immunotherapy. In this paper, expression, purification of human HSP70D and its adjuvant effect were studied.According to the cDNA sequence of human HSP70D, we designed primers to amplify full-length code region of human HSP70D. Upstream primer (5' TA CAT ATG GCC AAA GCC GCG GCA GTC G) contained start codon ATG and a Nde I site in 5' terminal; Downstream primer (5' TG CTC GAG ATC TAG CTC CTC AAT GGT GGG) without stop codon TGA contained a Xho I site in 5' terminal. The PCR product was designed to insert into pET-24a(+), by Nde I/Xho I double digestion. Secondly, full-length code region of HSP70D was amplified by RT-PCR from HeLa cells, a human cervix epitheloid carcinoma cell line. Total RNA of HeLa cells was extracted using Trizol reagent and 5 g total cellular RNA was reverse-transcribed to first strand cDNA by AMV reverse transcriptase. The synthesis of cDNA was checked by PCR with human p-actin primers. Then PCR with HSP70D upstream and downstream primers was subjected to denaturing(95 C for 15 seconds), annealing(59 C for 30 seconds), estension(72 C for 1 minute) for 25 cycles and 72 C for lOminutes using a Perkin-Elmer GeneAmp PCR system 9600. The PCR product contained 1933 bp, with Nde I and Xho I restriction siteson the 5' and 3' terminals. After PCR purification and Nde MXho I digestion, the fragment was ligated to pET-24a(+)and transformed into E. coli strain DH5a. Positively inserted clones were determined by Nde l/Xho I digestion and subjected to automated DNA sequencing with common primers T7 promotor and T7 terminator, and 4 gene specific primers. The correct recombinant was re-transformed into E. coli strain BL21[F-, ompT, hsdS(rB-, mB-), gal]. Positive clones were determined by Nde \IXho I digestion and designated as pET24a- HSP70D, which was used in the expression and purification of HSP70D fusion protein and its functional analysis.In order to get enough amount of recombinant human HSP70D for further clinical study, we optimize the procedure of fermentation of the engineered bacteria as well as purification of recombinant protein therein.By a small scale fermentation experiment, the M9CA plus medium were selected as the optimal growth medium. In our system, the recombinant protein expression was controled by Lac promoter, and can be induced by addition of low concentration of IPTG, typically, 0.1-5mM IPTG. The optimal concentration of IPTG and the induction time were determined to be ImM and 4 hours respectively.The rhHSP70D existed as soluble form in the bacteria cytosol. After disruption of the bacteria cells and centrifugation, the supernatant was collected and used as the starting material for further purification. Because of the high concentration of much hydrophobic contaminants existed, we used 20% ethanol precipitation for clarification of supernatants. The supernatant after ethanol precipitation became less viscosity and is suitable for subsequent manipulation with column chromatography. By using a sequence of ethanol precipitation, anion exchanger, and hydrophobicity interaction chromatography, and finally high resolution gel filtration chromatography, an electrophoretically pure rhHSP70D could be obtained. Subsequent high sensitivity gel immunoelectrophoresis proved the isolated protein to be free of bacteria protein. The Down-Stream- process was designed and optimi...