| Glyceraldehyde3phosphate dehydrogenase (GAPDH) was a kind of conservative andimportant multifunctional enzymes in the organisms. It was widely existed in prokaryote andeukaryote, such as Escherichia coli (E. coli), yeast, worms, rats and mice，etc. Previous studies havedemonstrated that GAPDH mainly worked in the sixth step of glycolysis in the glucose metabolismof the biological organisms. It could catalyze glyceraldehyde3phosphate to transfer to1，3-diphosphate glyceric acid and release energy ATP. The molecular weight of GAPDH monomer inthe biosphere was30kD-40kD，and its natural state was tetramers. The amino acid sequences ofGAPDH in prokaryote and eukaryote were highly conserved and the homology was as high as76%.GAPDH was a kind of important superfamily in prokaryote and eukaryote, and encoded bydifferent genes in different species, such as gapA, gapB and gapC genes in microbe，TDH-1，TDH-2and TDH-3genes in yeast, gpd-1，gpd-2，gpd-3and gpd-4genes in nematode and G3PDHgene in rat, etc. The fact that different coding genes in different species indicated the compensatingeffect of these genes and also indicated the high regulating effect of one species when they facedwith different environment stress. All of these provided a signiifcant reference for the tfinctionalconservation of GAPDH as a housekeeping protein. The internal standard protein was a kind of important housekeeping proteins in the organismsand had a higher sequence conservation and wide tissues distribution. There were a lot of internalstandard proteins according to some literatures reported, such as the the cytoskeleton protein P-actin,the microtubule protein Tubulin and the cell metabolism regulating protein GAPDH, etc. GAPDHhas been widely used for RT-PCR，Western blot and other molecular biology experimental as aninternal control due to its constant of the amount of protein expression in the same cell or tissue andrarely influenced by various stress factors. Although GAPDH was widely used as an internalstandard protein, no study was reported on its internal standard protein properties in prokaryote.Except for our previous study revealed GAPDH could act as an internal standard protein for therelative qualiifcation of prokaryotic proteins, there was no report on GAPDH as an internal standardprotein for the relative qualiifcation of prokaryotic proteins.In order to look for an internal protein for the relative quanliifcation of heterologous proteinsin the prokaryotic expression system, the different mouse derived monoclonal antibody was chosenaccording to its application in eukaryotic, such as P-actin, a-tubulin and GAPDH. Unexpextedly,mouse derived GAPDH monoclonal antibody could specilly recognized the GAPDH protein in E.coli BL21(DE3) cells and its expression level was stable and not alter along with inducing timeprolong. Therefore，we hypothesized that GAPDH could probably act as an internal standardprotein for prokaryote, and may be used as a universal internal standard protein for prokaryote andeukaryote.According to the aboved hypothesis, the GAPDH expression level of E. coli (includingBL21(DE3)，HB101，DH5a and OP50)，yeast, worms, PC12cells，mice and rats brain tissues undernormal conditions was examined using mouse derived GAPDH monoclonal antibody, and theGAPDH expression level of E. coli BL21(DE3), yeast, worms, PC12cells under different stressconditions was also explored. Results demonstrated that mouse derived GAPDH monoclonalantibody could widely recognize the GAPDH protein in different species, and there was differencebetweeb the content and molecular weight of GAPDH in different species. The molecular weight ofGAPDH in prokaryotes was lower than that of eukaryotes. Meanwhile, the GAPDH in differentspecies could relatively and steadily expressed in different stresses, such as the expression ofGAPDH in E. coli was not altered following with the increase of inducer IPTG and inducing timeprolong. The expression of GAPDH in yeast was not altered following with the increase of inducermethanol and inducing time prolong. The expression of GAPDH in C. elegans was not alteredfollowing with the increase of paraquat. The expression of GAPDH in PC12cell was not alteredfollowing with the increase of Sodium sulifte. All of these indicated that GAPDH could widely and steadily express in prokaryotes and eukaryotes, and could be used for a universal internal standardprotein for prokaryote and eukaryote.Furthermore, to conifrm which GAPDH version in prokaryotes could be recognized by themouse derived anti-GAPDH monoclonal antibody, the interaction protein of E. coli HB101wasprecipitated by Co-IP with the mouse derived anti-GAPDH monoclonal antibody followed withMALDI-TOF-MS identiifcation. Results demonstrated that GapA in E. coli HB101was recognizedby the mouse derived anti-GAPDH monoclonal antibody and the sequence coverage was52%.Simultaneously, to clarify the recognized character of the GAPDH version, the coding sequence ofvarious GAPDH genes in E. coli BL21(DE3), yeast GS115，C. elegans and PC12cells weresubcloned to the prokaryotic expression vector pET28b，and expressed by the inducer IPTG andidentiifed by Western blot. Results demonstrated that different GAPDH versions were highlyexpressed in prokaryotic expression system, and GapA in E. coli, Gap-3yeast GS115，Gpd-1,2，3，4in C. elegans and G3PDH in PC12cells were specially recognized by the mouse derivedanti-GAPDH monoclonal antibody, but not for GapC in E. collTo elucidate the bioactivity of over-expressed different GAPDH versions, the dynamic enzymeactivity of￡. coli cell lysate was determinated. Results demonstrated that the E. coli cell lysate withover-expressing different GAPDH versions had obvious enzyme activity, except for GapC in E. coli.These data indicated that GapA protein mainly involved in glycolysis of prokaryotes. Finally, thehomology of different GAPDH versions were analyzed by ClustalW/X sotfware and the evolutionof different GAPDH versions were also analyzed by the phylip-3.69sotfware. Results demonstratedthat the GAPDH genes of different species had a high sequence homology (>76%), and they had ahigh conservation during the evolution. However, the gapC gene in E. coli was far away, whichcould explain why the GapC protein had a lower enzyme activity and not recognized by the mousederived anti-GAPDH monoclonal antibody. Taken together, this study demonstrated that the mousederived anti-GAPDH monoclonal antibody could be used to determinate the universality and thestability of GAPDH under different normal and stress conditions, and tfirther conifrm GapA proteincould be used for the internal standard protein of prokaryotes. It provided a signiifcant reference forthe relative quantiifcation of prokaryotic and eukaryotic proteins.In addition, we have established two works associated with the research and development ofbiotechnology as following shown:1. To establish a novel method for tissue samples long-term stable storage and long-distancetransportation using rfeeze-drying technology: Lyophilization has been widely used for preservation,such as in food industry, pharmacy, biotechnology and tissues engineering，etc. However, there isno report on whether it could affect stability of RNA and protein levels in biological tissue samples. Herein we show that lyophilization can be used for storage of biological tissue samples without lossof bioactivities even stored at room temperature for7-14days. To address this issue, C57BL mousetissues were prepared and dried by lyophilization and a baking method, respectively, followed byexamination of morphological structure and total proteins by SDS-PAGE as well as gelatinzymography. Subsequently, the stability of RNAs and proteins, which were lyophilized and storedat room temperature (23°C) for14days was further examined by RT-PCR, SDS-PAGE and westernblot. Results demonstrated that lyophilization did not alter total protein activities of various tissues,including enzyme activities, immunoreactivities and phosphorylation, and did not affect severalRNAs in lyophilized tissues. Taken together, lyophilization may represent a valuable approach forpreservation and long-distance shipment of biological samples, particularly for the internationalexchange of biological samples without altering their bioactivities.2. To study the universality and the mechanism of Tat peptide to promote heterologous proteinhigh-yield and soluble expression in E. coli: As a cell-penetrating peptide, the Tat core peptide(YGRKKRRQRRR, pl=12.8) was coded by HIV-1virus and enriched with basic amino acid. Itcould deliver several heterologous macromolecules across biomembranes and without lose theirbioactivity. In our pilot study for exploring its transduction function, we found interestingly that ithad a novel function to promote the translation and expression of heterologous proteins in E. coli.Most suprisingly, we found that the Tat core domain was not only able to accelerate expression ofheterologous proteins in E. coli, but also prompted the yield and the solubility of heterologousproteins (5-10fold), however the mechanism was still unknown althouth we have published the firstreport on this issue. To address this issue, superoxide dismutase superfamily genes, includingSod-A，B，C inE. coli, Sod-1in Yeast, Sod-1，2，3，4，5in C. elegans, Sod-1in mouse were choseand subcloned to prokaryotic expression vectors pET28b-Tat and pET28b，and proteins wereinduced by isopropyl-|3-D-thiogalactopyranoside (IPTG) and equal amount proteins wererfactionated by15%SDS-PAGE. Subsequently, the yield of Sod-A, B，C in E. coli was identifiedby Western blot and the mRNA transcriptional level was also determined by real-time PCR.Beyond that, the total enzyme activity of Sods in E. coli cells was detected, and their bacterialanti-paraquat (PQ) activity and that of its mutation strains were also detected by microbial growthdetector. Expectedly, the yields and solubility of Tat tag proteins were higher than that of Tat freeproteins with induced time prolonging, and the enzyme activity of Tat tag proteins was also higherthan that of Tat rfee proteins. Real-time PCR results demonstrated that mRNA transcriptional levelof Tat tag proteins was higher than that of Tat rfee proteins. It suggested that Tat core peptide couldincrease mRNA transcriptional level to promote heterologous proteins high-yield and solubleexpression in E. coli. Furthermore, bacterial which was over-expressed Tat-SodA and SodA had along lifespan compare with control, and Tat-SodA also had longer lifespan in contrast to that ofSodA, and SodA mutation strain had lower lifespan than WT, but Sod-B, C mutation strains did not.Taken together, Tat core peptide as a significant basic peptide could increase mRNA transcriptionalto promote heterologous proteins SodA, B，C high-yield and soluble expression in E. coli and further had a signiifcant resistance to4mM PQ via SodA. This study provided a signiifcanttechnique for heterologous proteins high-yield and soluble expression in E. coli and would bewidely used in biotechnology and bioengenring fields. |
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