Pif1 subfamily helicase is conserved from yeast to humans with a lot of cellular functions. In order to elucidate the function of human PIF1 helicase from biochemical level, we cloned human PIF1 gene by PCR from HeLa cell cDNA library. We co-transformed a pMStRNA1 plasmid encoding rare tRNA codons and a plasmid encoding molecular chaperon to greatly enhance the overexpression of human PIF1 protein. Finally we purified full-length PIF1 helicase by column chromatograph carried out at 4 degrees C using fast protein liquid chromatograph (FPLC) system. The human PIF1 protein was purified in enough quantity for detailed biochemical analysis. Biochemical assay showed that PIF1 had ATPase activity and helicase activity. The purification and biochemical properties analysis of human PIF1 helicase will allow us to understand how, at the molecular and mechanistic level, this conserved helicase operates in the cell.
DNA Helicases ; biosynthesis ; genetics ; metabolism ; HeLa Cells ; Humans ; RNA, Transfer ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

Human DNA Topoisomerase I (hTopo I) has been identified to be an efficient target of many effective antitumor drugs. Natural hTopo I is not convenient to be used in screening because of its low concentration in cells. In order to fast screen new anticancer drugs targeting at hTopo I from natural compounds in vitro, hTopo I gene open reading frame (ORF) has been successfully cloned and overexpressed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo I specific antisense primer and the hTopo I ORF was synthesized by PCR. After digestion with EcoR I and Kpn I, the synthesized fragment was inserted into pPICZaA, gave rise to pPICZalpha-hTopoI. After digestion with Sac I, the lined pPICZalpha-hTopoI was transformed into Pichia pastoris strains (KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000 ug/mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD-hTopol) could overexpress recombinant hTopo I, which was fused to the alpha-factor secretion signal and could be secreted into the supernatant in the culture. alpha-factor could be cleaved from the expressed protein during secretion. A higher activity amount of the enzyme was secreted by the particular strain SMD-hTopoI because of its absence of proteimase A than by other strains which possess proteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-hTopoI was induced in BMMY (pH7.25) at 20 degrees C , with addition of 0.5% (V/V) methanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 43 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protein in the culture supernatant. SDS-PAGE and Western blot analyses showed that the mass of the recombinant hTopo I was 91 kD with no glycosylation.
DNA Topoisomerases, Type I ; biosynthesis ; genetics ; Fermentation ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Human manganese superoxide dismutase (hMn-SOD) cDNA was amplified by RT-PCR from total RNA of human liver cell (L02), and cloned into yeast expression vector pPIC9K containing AOX1 promoter and the alpha-factor signal peptide sequence. The resultant pPIC9K-MnSOD was transformed to P. pastoris GS115, screened for Mut+ carrying multiple copies of hMn-SOD. The positive transformants were fermented in flasks and induced by 0.5% methanol. After 4 days of methanol induction, the expressed hMn-SOD was up to 32% of the total proteins in the supernatant by SDS-PAGE with specific activity of 247.7 u/mg.
Cloning, Molecular ; DNA, Complementary ; biosynthesis ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Superoxide Dismutase ; biosynthesis ; genetics

In 2010, the artificial synthesis of Mycoplasma mycoides triggers the new era of synthetic biology. This great breakthrough is achieved mainly thanks to the powerful DNA recombinant ability of yeast. In recent years, except for the methods used for large DNA assembly on the basis of in vivo homologous recombination, various different DNA assembly methods in vitro, based on the concept of DNA ligation or polymerization, have also been developed, such as Biobrick\BglBrick, SLIC and Gibson one-step assembly. Application of these new technologies has greatly accelerated the construction of synthetic part libraries, biosynthetic pathway and even microbial chromosomes. In fact, all DNA assembly methods are derived from the combinations of DNA joining and organizational schemes. This review describes the brief introduction of the main in vivo and in vitro DNA assembly protocols developed so for, which will benefit the construction of different types of synthetic functional devices and also biosynthetic pathways in the research of synthetic biology in China.
DNA ; biosynthesis ; chemistry ; genetics ; DNA, Recombinant ; biosynthesis ; genetics ; Genetic Engineering ; methods ; Metabolic Networks and Pathways ; Synthetic Biology ; methods

OBJECTIVETo express a multi-copy specific epitope recombinant protein of herpes simplex virus type 1 (HSV-1) with immuno-reactivity.

METHODSMulti-copy genes with a specific epitope of HSV-1-glycoprotein G 112-127 were constructed by DNA recombination and cloned in E. coli JM109 pGEM-5Zf. The positive recombinants were determined by SDS-PAGE and Western blotting.

RESULTSThe recombinants with 4, 8, 16 and 32 copies of gG 112-127 were obtained. The 8-copy recombinant was expressed by 17.5%, mainly as inclusion body. And it reacted with antiserum HSV-1, but not with antiserum HSV-2.

CONCLUSIONThe HSV-1-gG112-127 recombinant could be used to distinguish HSV-1 and HSV-2 in ELISA.

Antigen-Antibody Reactions ; DNA, Recombinant ; Epitopes ; biosynthesis ; immunology ; Herpesvirus 1, Human ; immunology ; Herpesvirus 2, Human ; immunology ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; Viral Envelope Proteins ; biosynthesis ; immunology

Ranpirnase (onconase, ONC) is a new drug, with weak RNase activity and strong cytotoxicity to various tumor cells in vitro and in vivo. This study is to obtain recombination onconase (rONC) with high bioactivity. Based on the codon preference of Pichia pastoris, we designed and synthesized the gene according to cDNA sequences of ONC and the α mating factor's prepeptide. We screened positive clones after transforming the recombination plasmids into P. pastoris X-33, GSS115 and SMD1168. We screened the best combination of seven different vectors and host strains. Moreover, we optimized culture condition in shake flasks and 10 L bioreactor, and purified rONC from the supernatant after inducing it with 0.25% methanol by aqueous two-phase extraction coupling G50 molecular exclusion method. The highest rONC production was 13 mg/L in pPICZα-A/X-33/ONC combination under the condition of pH 5.5 and 23 degrees C in shake flasks for 7 d; and that the highest rONC production was 180 mg/L when the induction is performed in the lower basic salt medium with pH 5.5 in the 10 L bioreactor for 7 d. The yield of rONC is more than 90% at a purity of above 95%. rONC can kill various tumor cells in vitro. The expression and purification of rONC would be useful for further investigation of this new drug.
Antineoplastic Agents ; metabolism ; Bioreactors ; Cell Line, Tumor ; Codon ; DNA, Complementary ; Genetic Vectors ; Humans ; Pichia ; metabolism ; Recombinant Proteins ; biosynthesis ; Ribonucleases ; biosynthesis

OBJECTIVETo clone and express the HIV-1B gp120 genes isolated at different organizations from a patient died of AIDS dementia complex (ADC) in eukaryotic cells.

METHODSUsing the genomic DNA isolated from peripheral lymphnodes, choroid plexus and occipital white matter from a patient died of ADC as the template, HIV-1B gp120 gene was amplified with PCR. After sequenced, HIV-1B gp120 was inserted into pcDNA3.1 (+) and recombinant expressing vector gp120/pcDNA3.1 (+) was constructed succeffuly confirming with sequencing. Then expressing vector was transfected into eukaryotic cells U87 using liposome transfection and expression of HIV-1B gp120 gene was assayed with indirect immunofluorescence.

RESULTSHIV-1B gp120 genes isolated from peripheral lymphnodes, choroid plexus and occipital white matter of the ADC patient were successfully cloned and recombinant expressing vector gp120/pcDNA3; 1 (+) could express envelope glycoprotein HIV-1B gp120 in U87 cells.

CONCLUSIONAll the HIV-1B gp120 gene isolated at the different organizations of the same ADC patient could express in U87 cells, which may supply a valuable basis for studying the neurotoxicity and neurotoxic mechanism of HIV-1 gp120 protein.

AIDS Dementia Complex ; virology ; Cloning, Molecular ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; toxicity ; Humans ; Recombinant Proteins ; biosynthesis ; Sequence Analysis, DNA

Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
Animals ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Fireflies ; enzymology ; Luciferases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29 degrees C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163+/-4) U/(mgmin), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.
Antioxidants ; pharmacology ; DNA, Complementary ; Electroporation ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; Serpins ; biosynthesis ; genetics

OBJECTIVETo construct the recombinant adenovirus vector expressing human endostatin.

METHODSHuman endostatin gene extracted from pGEM-T Easy vector containing the target gene fragment was successfully amplified using PCR and cloned into pShuttle2 vector. The target gene was subcloned into an adenovirus vector and the resulted recombinant adenovirus (Ad-hEndo) was linearized before transfected into HEK 293 packaging cells. The Ad-hEndo recombinant adenovirus was efficiently amplified in 293T cells and purified by CsCl density centrifugation, and the titer of the virus was determined.

RESULTSThe amplified hEndostatin cDNA was verified by PCR and sequencing, and the resulted virus titer reached 5.2 x 10(9) pfu/ml.

CONCLUSIONThe recombinant adenovirus containing human endostatin gene has been successfully constructed, which may provide important basis for gene therapy research for angiogenesis-dependent diseases.

Adenoviridae ; genetics ; Cell Line ; Cloning, Molecular ; DNA, Recombinant ; genetics ; Endostatins ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection