Rhizopus oryzae lipase (ROL) is not only a biocatalyst used in a broad range of biotechnological fields, but also a model to investigate the function of intramolecular chaperone in the post-translational processing of lipase. In this study, we cloned and expressed the mature lipase gene (m-ROL) containing the pre-sequence (pro-ROL) of R. oryzae HU3005 in Pichia pastoris GS115 and characterized their enzymatic activities. m-ROL exhibited higher hydrolysis activity towards middle-chain substrates (C10 and C12) at pH 9.0, whereas pro-ROL preferred short-chain substrates (C4) and displayed maximal activity at pH 8.0. Moreover, pro-ROL possessed better thermal stability than m-ROL. This enzymatic discrepancy between m-ROL and p-ROL may be due to the pre-sequence that affects the folding and conformation of the mature lipase domain. To improve the expression level of m-ROL in P. pastoris, overlap extension PCR was conducted to substitute eight less-frequently used codons of m-ROL with frequently used codons. After methanol-induced expression for 72 h, the activity and protein content of the codon optimized m-ROL reached 132.7 U/mL and 50.4 mg/L, while the activity of the parental m-ROL and pro-ROL are 28.7 U/mL and 14.4 mg/L, 29.6 U/mL and 14.1 mg/L, respectively.
Codon ; Enzyme Precursors ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; Lipase ; biosynthesis ; genetics ; metabolism ; Pichia ; enzymology ; genetics ; Protein Engineering ; methods ; Protein Folding ; Recombinant Proteins ; biosynthesis ; genetics ; Rhizopus ; enzymology ; genetics ; Substrate Specificity

Hepatitis B virus X protein (HBx) has various functions and plays a crucial role in the development of hepatocellular carcinoma (HCC). However, due to different transfection efficiency levels and experimental approaches, it is difficult to correlate the exact functions of HBx to HBV-associated HCC. In this study, we constructed two prokaryotic expression vectors, pGEX-HBx-EGFP-TLM and pGEX-EGFP-TLM, which expressed HBx-EGFP-TLM and EGFP-TLM fusion proteins respectively. Both vectors contained a coding sequence of TLM transduction motif derived from the PreS2-domain of Hepatitis B Virus surface antigens. In addition, EGFP was expressed as a reporter reflecting the transduction efficiency of TLM. The fusion protein HBx-EGFP-TLM or EGFP-TLM purified from Escherichia coli BL21(DE3) by AKTA Purifier system was incubated with AML12 and SMMC-7721 cells. Both Western blotting and laser confocal results indicated that the translocation motif TLM could lead HBx-EGFP and EGFP into the cytoplasm. Dual-Luciferase Reporter Assay revealed that the activity of mEZH2 promoter could be up-regulated by the recombinant HBx. In conclusion, we expressed a cell-permeable HBx, which could provide a new method to study the functions of HBx.
Amino Acid Motifs ; genetics ; Cell-Penetrating Peptides ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hepatitis B Surface Antigens ; genetics ; Protein Precursors ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Trans-Activators ; biosynthesis ; genetics

Chymosin is an important industrial enzyme widely used in cheese manufacture. To improve expression efficiency of recombinant bovine chymosin in Kluyveromyces lactis strain GG799, we designed and synthesized a DNA sequence encoding bovine prochymosin gene (GenBank Accession No. AA30448) by using optimized codons. The synthesized prochymosin gene was amplified by two-step PCR method, and then cloned into the expression vector pKLAC1, resulting in pKLAC1-Prochy. pKLAC1-Prochy was linearized and transformed into K. lactis GG799 by electrotransformation. Positive clones were screened by YEPD plates containing 1% casein. A recombinant strain chyl with highest activities and multi-copy integration which was detected by using specifical integration primers was chosen and fermented in flask. Prochymosin was expressed in K. lactis successfully. SDS-PAGE analysis revealed that the purified recombinant bovine prochymosin had a molecular mass of 41 kDa. After acid treatment, molecular weight of chymosin is about 36 kDa, the same as native bovine chymosin. Activity tests showed that the chymosin activity of the culture supernatant was 99.67 SU/mL after 96 h cultivation. The activities of chymosin were not prominent increased when galactose was used as carbon source instead of glucose, which proved that the fermentation of recombinant strain does not need galactose inducing. The recombinant K. lactis strain obtained in this study could be further used to produce recombinant chymosin for cheese making.
Animals ; Cattle ; Chymosin ; biosynthesis ; genetics ; Enzyme Precursors ; biosynthesis ; genetics ; Gene Expression Regulation, Fungal ; genetics ; Genetic Vectors ; genetics ; Kluyveromyces ; genetics ; growth & development ; metabolism ; Protein Engineering ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo construct a yeast expression vector of hepatitis B virus (HBV) PreS1 gene using the Sos-recruitment system (SRS), and evaluate the effect of the expression product on the growth of the yeast cells and activation of the reporter gene.

METHODSThe coding sequence of HBV preS1 was amplified by PCR and cloned into the yeast expression plasmid pSos. The recombinant bait plasmid pSos- PreS1 was verified by sequencing before transformation into competent yeast cells. The effects of the expression product on the yeast cell growth and activation of the reporter gene were evaluated.

RESULTSThe yeast expression vector of HBV PreS1 gene was constructed successfully. The recombinant bait plasmid showed no toxic effect on yeast cdc25H cells without a self-activation of the reporter gene.

CONCLUSIONThe SRS can be used to study the proteins interacting with HBV PreS1 protein and provides a means for obtaining insight into the pathogenic mechanism of HBV.

Cloning, Molecular ; Genetic Vectors ; genetics ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; Humans ; Plasmids ; genetics ; Protein Precursors ; biosynthesis ; genetics ; Receptors, Virus ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Two-Hybrid System Techniques ; Yeasts ; genetics ; metabolism

Nerve growth factor (NGF) promotes neuronal survival and differentiation and stimulates neurite outgrowth. NGF is synthesized as a precursor-proNGF in vivo. In this paper, a pET-proNGF prokaryocyte expression vector was constructed and transformed into E. coli BL21(DE3)pLysS. The proNGF was expressed in the form of non-active aggregated monomer in E. coli after induction with IPTG. SDS-PAGE revealed the proNGF expression product had a Mr.30.2 kD. Western blotting analysis showed that the protein had good antigenicity. Fusion protein was successfully purified by Ni2+-NTA affinity chromatography and cleaved by Enterokinase and 13.1 mg proNGF was obtained from 100 mL cell culture in a typical experiment. The protein was dialyzed in a redox system containing reduced and oxidized glutathione. RP-HPLC was used to analysis the result of the refolding. The refolded proNGF protein can induce neurite outgrowth of PC12 cells, which indicated that pro-form of NGF we obtained had biological activity.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Humans ; Nerve Growth Factor ; biosynthesis ; genetics ; Protein Precursors ; biosynthesis ; genetics ; Protein Renaturation ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

The preparation process and immunogenicity of a novel hepatitis B vaccine containing preS1, preS2 and S epitopes were investigated in this study. A Pichia pastoris stain GS115-SS1S2 harbouring two chimeric HBsAg gene constructs, SS1 and SS2 was cultivated by high-density fermentation. 300-600 mg/L of the expression level was achieved through 48-72 h methanol induction. SSIS2 antigen was extracted and purified by silica adsorption, HIC and SEC to 99% purity from the harvested cells. 82 mg purified antigen could be achieved from one liter of fermentation culture. The immunogenicity of the purified antigen was evaluated in NIH mice. Three groups of female NIH mice, 14-16 g in weight, were injected once intraperitoneally with 2.5, 0.625, 0.156 microg of each of the two vaccines: SS1S2 or a commercially available S vaccine. Part of the mice were bled in 30 days after injection to compare the ED50 of the two vaccines. For the SSIS2 vaccine, the ED50 is 0.46, 0.29 and 0.84 microg respectively for the preS1, preS2 and S antigens. For the S vaccine, the ED50 is 0.99 microg for the S antigen. Another part of the mice were bleed in 7 or 14 days to detect preS1, preS2 and S antibodies. Higher ratios of mice were seroconverted for preS1 and preS2 antibodies as compared to the S antibody in these two time points. These results suggest that the SS1S2 vaccine may be more immunogenic than the conventional S vaccines.
Animals ; Epitopes ; immunology ; Female ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; biosynthesis ; immunology ; Mice ; Pichia ; genetics ; metabolism ; Protein Engineering ; Protein Precursors ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Envelope Proteins ; immunology

BACKGROUNDThe hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein.

METHODSThe reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis.

RESULTSForty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class II lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 (LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327).

CONCLUSIONSGenes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S1 protein.

Amino Acid Sequence ; Base Sequence ; DNA, Complementary ; chemistry ; Gene Library ; Hepatitis B Surface Antigens ; genetics ; physiology ; Humans ; Leukocytes ; metabolism ; Molecular Sequence Data ; Plasmids ; Protein Interaction Mapping ; Protein Precursors ; genetics ; physiology ; Recombinant Fusion Proteins ; biosynthesis ; Transcriptional Activation ; Two-Hybrid System Techniques ; Yeasts ; genetics

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.
Apolipoprotein A-I ; biosynthesis ; genetics ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

The Pichia pastoris strain GS115-PreS could produce a high expression level of full-length PreS protein that secreted to the supernatant after methanol induction in the fermentation. The Western blot analysis showed a single band with expected molecular mass of 48kD and that the major component of the particles was the full-length PreS protein (PreS1 + PreS2 + S) and small envelope protein (S) of 48 and 28 kD, respectively. Electron microscopy image showed PreS particles with 30 nm in diameter. The supernatants of the fermentation were desalted and concentrated. Purified PreS protein was obtained by DEAE-SFF anion exchange column chromatography and the PreS particles were obtained by ultracentrifugation and sucrose density gradient. The ELISA assay results proved that both full-length PreS protein and particles showed high immunogenicity and specificity. P/N ratio further demonstrated that the immunogenicity of the particles is higher than the full-length PreS protein.
Hepatitis B Surface Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Viral Envelope Proteins ; biosynthesis ; genetics

OBJECTIVETo evaluate the effects of acute glucose level changes on expression of prepro-orexin, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) mRNA in rat hypothalamus tissue and pancreatic islets cells.

METHODSThirty adult male Wistar rats were randomly divided into three equal groups (n = 10). The acute hypoglycemia rat model was induced by a single subcutaneous injection of insulin. Twenty acute hypoglycemia rats were divided into group B and group C. Group B was allowed to eat freely, while group C was food-deprived. Control rats were injected the same volume of saline. The effect of glucose levels (2.8 mmol/L and 8.3 mmol/L) on pancreatic islet cell orexin system was detected in pancreas islet cell cultured in vitro. The expression of prepro-orexin and OXR mRNA was examined in rat hypothalamus tissue and pancreatic islets cell cultured in vitro using reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSExpression of orexin mRNA increased about 150% for the food-deprived hypoglycemia rats in comparison with control group (P < 0.01), whereas expression of OX1R mRNA decreased up to 30% (P < 0.01). However, expression of OX2R mRNA was unchanged in comparison with control group. In vitro, after incubation with 2.8 mmol/L glucose for 6 hours, the expression of prepro-orexin mRNA increased 2 times in rat pancreas islet cells in comparison with 8.3 mmol/L glucose group (P < 0.01). But the expression of OX1R mRNA was not sensitive to acute glucose fluctuation.

CONCLUSIONSOrexin in rat hypothalamus is stimulated by decline in blood glucose and inhibited by signals related to feeding. Moreover, glucose plays a role in modulating the gene expression of prepro-orexin in rat pancreatic islet cells.

Animals ; Blood Glucose ; metabolism ; Glucose ; pharmacology ; Hypoglycemia ; metabolism ; Hypothalamus ; metabolism ; Insulin ; pharmacology ; Intracellular Signaling Peptides and Proteins ; genetics ; Islets of Langerhans ; metabolism ; Male ; Neuropeptides ; biosynthesis ; genetics ; Orexin Receptors ; Orexins ; Protein Precursors ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Wistar ; Receptors, G-Protein-Coupled ; Receptors, Neuropeptide ; biosynthesis ; genetics