Analysis of the limitations of hepatitis B surface antigen expression in soybean cell suspension cultures.

Soybean cell suspension cultures were transformed using Agrobacterium tumefaciens harboring pHBS/pHER constructs to express hepatitis B surface antigen (HBsAg). The transformed colonies were selected and analyzed for the expression of HBsAg by PCR, reverse transcription (RT) PCR, Western blot and ELISA analysis. The maximum expression of 700 ng/g F.W. was noted in pHER transformed cells. The highest expressing colonies were used to initiate the cell suspension cultures and the expression of HBsAg was estimated periodically. The expression levels were reduced drastically in cell suspension cultures compared to the colonies maintained on semi-solid medium. Various parameters were studied to maximize the cell growth and to retain the expression levels. The supplementation of culture medium with a protease inhibitor, leupeptin hemisulfate could restore up to 50% of HBsAg expression in cell suspension cultures. This is the first report to investigate the possible cause and solution to the loss of recombinant protein expression levels in plant cell suspension cultures.

Secretion of hepatitis B surface antigen in transformed tobacco cell suspension cultures.

Six different expression cassettes of hepatitis B surface antigen (HBsAg) were used to transform tobacco cell suspension cultures. The transgenic nature of the cells was confirmed by PCR. The secreted HBsAg was assayed by ELISA and analyzed by Western blotting. A maximum of 31 microg antigen/l was obtained in the spent medium from the transformed cells. The use of an ethylene-forming enzyme promoter and incorporation of C-terminal endoplasmic-reticulum-retention signal enhanced the secretion of HBsAg. Salicylic or jasmonic acid at 10 microM: increased secretion of HBsAg by six fold.

Expression of hepatitis B surface antigen in transgenic banana plants.

Embryogenic cells of bananan cv. Rasthali (AAB) have been transformed with the 's' gene of hepatitis B surface antigen (HBsAg) using Agrobacterium mediated transformation. Four different expression cassettes (pHBS, pHER, pEFEHBS and pEFEHER) were utilized to optimize the expression of HBsAg in banana. The transgenic nature of the plants and expression of the antigen was confirmed by PCR, Southern hybridization and reverse transcription (RT)-PCR. The expression levels of the antigen in the plants grown under in vitro conditions as well as the green house hardened plants were estimated by ELISA for all the four constructs. Maximum expression level of 38 ng/g F.W. of leaves was noted in plants transformed with pEFEHBS grown under in vitro conditions, whereas pHER transformed plants grown in the green house showed the maximum expression level of 19.92 ng/g F.W. of leaves. Higher monoclonal antibody binding of 67.87% of the antigen was observed when it was expressed with a C-terminal ER retention signal. The buoyant density in CsCl of HBsAg derived from transgenic banana leaves was determined and found to be 1.146 g/ml. HBsAg obtained from transgenic banana plants is similar to human serum derived one in buoyant density properties. The transgenic plants were grown up to maturity in the green house and the expression of HBsAg in the fruits was confirmed by RT-PCR. These transgenic plants were multiplied under in vitro using floral apex cultures. Attempts were also made to enhance the expression of HBsAg in the leaves of transgenic banana plants by wounding and/or treatment with plant growth regulators. This is the first report on the expression of HBsAg in transgenic banana fruits.

Expression of hepatitis B surface antigen in tobacco cell suspension cultures.

Hepatitis B virus ' s ' gene coding for surface antigen was cloned into plant transformation vectors pHER100 and pHBs100 with and without endoplasmic reticulum retention signal, respectively. Transformed tobacco cell lines were analyzed for the integration of the transgene by PCR and Southern blot hybridization. Expression levels as determined by ELISA showed maximum expression levels of 2 microg HBsAg gm(-1) fresh weight and 10 ng mL(-1) of spent medium in pHER100 transformed cells. Western blot analysis confirmed the presence of 24 kDa band specific to HBsAg in the transformed cells. HBsAg was expressed both as intracellular and secreted forms in pHER100 transformed cells. The buoyant density in CsCl of HBsAg derived from pHBs100 transformed tobacco cells was determined and found to be 1.095 g mL(-1). HBsAg obtained from transformed tobacco cells is similar to the human serum derived one in buoyant density properties. This is the first report on the secretion of HBsAg particles by plant cells into the cell culture medium.

Identification and characterization of promoter elements responsible for the induction of the albumin gene by heat shock in early embryonic rat liver.

We had reported earlier that the expression of albumin increases upon heat shock in embryonic rat liver cells at about 12-13 days of gestation. Here, we report on the identification of heat shock elements (HSEs) within -450 bp of the rat albumin promoter using chloramphenicol acetyl transferase (CAT) assays done with the extracts from H4II-E-C3 cells transfected with plasmids carrying the CAT reporter gene under the control of different deletion fragments of the rat albumin promoter. Gel retardation assays done with synthetic oligonucleotides representing putative HSEs in the rat albumin promoter and H4II-E-C3 cell extracts show that the heat shock factors bind this region in a sequence-specific and reversible manner. Super-shift assays demonstrated that the HSEs present in the rat albumin promoter are bound by HSF1 and not by HSF2. This effect of heat shock on the expression of rat serum albumin is seen only in the liver and is not observed in other tissues, suggesting that HSF-mediated activation of albumin gene cannot overcome the negative regulatory factors present in other tissues. In addition to the HSEs, we have identified a putative GAGA factor binding site in the rat albumin promoter at -228 bp to -252 bp position. These GAGA repeats are bound in a sequence-specific and reversible manner by two factors in a nonstressed cell, whereas only one of these two factors continues to bind the GAGA repeats under heat shock conditions. The physiological significance of these results is discussed.

Differentiation of mouse embryonal carcinoma cells PCC4 by heat shock and the kinetics of induction of heat shock proteins.

Heat shock to embryonal carcinoma cells PCC4 at 45 degrees C for 30 min resulted in the differentiation of cells although heat shock response was induced on exposure to 42 degrees C for 60 min. Differentiated cells were large and well spread with reduced nuclear/cytoplasmic ratios as compared to undifferentiated cells. Change in cell morphology was associated with the disappearance and appearance of stage specific embryonic antigens 1 and 3 respectively. We also found a change in intracellular pH in PCC4 cells within 30 min of heat shock as measured by the change in fluorescence intensity of a probe incorporated into cells during heat shock.

Altered expression of alpha-fetoprotein and albumin in early embryonic rat liver upon heat shock.

During rat liver development alpha-fetoprotein (AFP) and albumin (alb) are differentially regulated. Alpha-fetoprotein synthesis starts soon after fertilization whereas the earliest time albumin could be detected is day 17-18. However during normal development trace amounts of albumin mRNA could be detected on day 14 or 15. When 12-13 day old embryonic rat liver cells are given heat shock at 42 degrees C, there is an enhanced expression of albumin mRNA as well as protein. Under heat shock conditions, unlike albumin mRNA, there is specific degradation of alphafetoprotein mRNA. Western blot analysis indicated that AFP that was already present is not affected by heat shock whereas alpha-fetoprotein mRNA is degraded.

Change in membrane organization induced by heat shock.

In vivo change in membrane organization in adult rat liver cells induced by heat shock was studied by incorporating the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) in these cells. DPH was essentially localized in the plasma membrane, as indicated by quenching of its fluorescence (over 90%) by 2,4,6-trinitrobenzenesulfonate (TNBS). Measurements of both fluorescence polarization and intensity indicate that there is an immediate change in membrane organization (order) following heat shock. The polarization changes of DPH and TMA-DPH incorporated into liver cell plasma membranes upon heat shock are in opposite directions. This result is attributed to the different locations of these probes in the membrane. This change in membrane organization could be a part of a general stress response which is then transmitted to other cellular organelles.

Characterisation of a high-molecular-weight developmentally regulated adult rat liver-specific protein.

A major, hitherto unknown protein which is shown to be developmentally regulated has been isolated from rat liver. The protein has a molecular mass of 150 kDa and constitutes 8-10% of the adult liver protein. Immunoblot experiments and immunofluorescence studies established its presence specifically in adult liver cells. This protein is not present in early embryonic liver cells, gets induced around day 18 of gestation and reaches near adult levels by 1 day after birth and is not found in the cells of an ascitic hepatoma. Unlike albumin and alpha-fetoprotein, the two well known developmentally regulated liver proteins, the 150 kDa protein is not secreted. Some of the characteristics of this liver-specific protein are discussed.

Heat-induced expression of albumin during early stages of rat embryo development.

An examination of heat-induced expression of proteins in tissues from adult and embryonic liver in rats shows that albumin, which is constitutively expressed in adult liver and is not synthesized in embryos before 16 days of gestation, appears in liver cells at earlier stages of development upon heat shock. On the basis of available evidence for the expression of heat shock proteins at distinct stages of development and on the basis of our findings, it may be argued that there could be common molecular events taking place during development and as a result of heat shock. We suggest also that one of the consequences of heat shock could be an internal change of pH within the cell which, in turn, might trigger alterations in gene expression.