Introduction

The human PNAS-4 gene is said to encode a new pro-apoptotic protein which was up regulated at the time of early response to DNA damage. There was significant cell death observed in U2OS cells, when hPNAS-4 is overexpressed in them. The hPNAS-4 expression significantly reduces the growth of tumor In Vivo and increases apoptosis of tumor cells. The tumor cells could experience reduced angiogenesis which indicates that hPNAS-4 is likely to be used effectively in a novel gene therapy for human cancer.


The current study deals with the purification, characterization, subcellular localization and polyclonal antibody production of hPNAS-4 protein. The purified hPNAS-4 protein and the synthesis of polyclonal antibodies are powerful tools for studying the apoptotic mechanisms of hPNAS-4.

Methodology

Plasmids

The expression plasmids pGEX-6p-I and pEGFP-NI of E.coli are purchased and cultured in LB medium supplemented with 100 micrograms/ml of ampicillin at 37 degree C. The human non-small cell lung cancer cell-line A549 was also obtained. Three types of recombinant plasmids were used in the experiment. The plasmid pGEX-hPNAS-4 was utilized to express glutathione transferase (GST) tagged hPNAS protein in E.coli. The plasmid pEGFP-hPNAS-4 was utilized to identify the localization of hPNAS-4 in mammalian cells. The recombinant plasmid pcDNA3.1 hPNAS-4 was expressed in eukaryotes where hPNAS-4 cDNA was included in the pcDNA3.1 vector.

The total RNA was obtained from human A549 cells. One more recombinant plasmid pEGFP-hPNAS-4 was made with the help of fluorescent EGFP or enhanced green fluorescent protein.

Expression and purification of recombinant protein


The recombinant plasmid pGEX-hPNAS-4 with GST tag was transferred into E. coli BL21 (DE3) cells. The transformants of E. coli BL21 (DE3) cells consisting of pGEX-hPNAS-4 were supplied with 0.5mM IPTG. Cell pellets are collected after centrifugation and were resuspended in lysis buffer. Later, the cells were disturbed by sonication and were centrifuged for separating soluble and insoluble fractions. The solubility state of the recombinant GST tagged hPNAS-4 protein in E. coli was checked by collecting the supernatant and cell pellets analyzed separately using SDS-PAGE.

The recombinant hPNAS-4 protein that is GST tagged was purified with Sepharose affinity chromatography. The soluble fraction was pushed into pre equilibrated glutathione sepharose 4B column. The eluded substance called SI was again loaded into Q-Sepharose fast flow column preequilibrated with buffer B. The hPNAS-4 protein tagged with GST were separated sequentially using buffer B mixed along with various concentrations of NaCl. The purity of protein was evaluated by densitometry of polyacrylamide gels stained with Coomassie Brilliant Blue. Protein concentration was identified by Spectrophotometer.

Western blot analysis was performed to detect the purified hPNAS-4 protein tagged with GST and crude recombinant hPNAS-4 using anti-GST antibody. The recombinant protein that is purified is detected by liquid chromatography followed by electrospray ionization -MS. The recombinant hPNAS-4 protein that is purified was utilized as immunogen to generate polyclonal antibodies in white rabbits of New Zealand. The titre of the antibody was evaluated by ELISA method. The polyclonal antibody specificity was examined by western blotting technique. The localization of hPNAS-4 in the cells is determined by the expression of the recombinant plasmid pEGFP-hPNAS-4 fused with EGFP epitope.

The constructed plasmid pcDNA3.1-hPNAS-4 was transfected to A549 cells to investigate the apoptosis status of the cells by Hoechst 33258 staining, DNA ladder electrophoresis and flow cytometry.

Results of the study

Expression

The recombinant hPNAS-4 protein tagged with GST was expressed approximately 40 to 50 percent in soluble form out of the total expression of hPNAS-4 protein. Hence, the soluble supernatant was used for further purification.

Purification

Glutathione-Sepharose affinity resin was used for purifying the recombinant protein to its homogeneity. The amount of purified recombinant hPNAS-4 protein obtained from 1 liter of bacterial culture was 9.78mg. The purification of the recombinant protein was improved by anion exchange procedure. The resultant purified recombinant hPNAS-4 protein appeared as single band on SDS-PAGE with 96 percent purity.

Identification of purified hPNAS-4

IPTG induced and uninduced cell lysates with hPNAS-4 protein tagged with GST were analyzed in western blotting using mouse anti-GST monoclonal antibodies. The reaction of these antibodies with the protein in uninduced cells was not observed while hPNAS-4 protein tagged with GST from induced cells could react with the antibodies.

The ESI-MS analysis revealed the presence of peptide peaks at m/z 883.705, 940.381 and 1083.420 matching the trypsin-digested peptide sequences of hPNAS-4 protein. The matched peptide sequence coverage was 26 percent.

Specificity of polyclonal antibodies for hPNAS-4

The polyclonal antibodies that are synthesized were able to react with hPNAS-4 protein tagged with GST. In western blot analysis, a single band of nearly 47 kDa was formed by the E.coli cell lysates induced by IPTG. The endogenous hPNAS-4 protein from A549 cells could react with polyclonal antibodies forming a band of 21 kDa.

hPNAS-4 localization

This protein was found to be present in perinuclear compartment and cell cytoplasm as there was brown staining in these regions. The staining is seen in A549 cells overexpressing hPNAS-4 protein.

Impact of hPNAS-4 on cell apoptosis

Higher expression of RNA was observed in A549 cells transfected with recombinant protein pcDNA3.1-hPNAS-4. The transfection of this recombinant protein would induce apoptosis of the cell. Hoechst 33258 staining showed the formation of condensed and bright blue cell nuclei in cells overexpressing pcDNA3.1-hPNAS-4. Flow cytometric analysis showed 36.9 percent of cells transfected with recombinant protein.

Reference:

Hongxin Deng, Qingyuan Jiang, Shufang Liang, Fei Yan, Shengyan Hou, Zhiyong Qian, Jiong Li, Yanjun Wen, Jinliang Yang and Yuquan Wei. Prokaryotic expression, purification, and characterization of a novel pro-apoptosis protein hPNAS-4. Biotechnol.Appl.Biochem. (2010) 55, 63-72. DOI: 10.1042/BA20090111.

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