The technology of genetic engineering has been widely used to express macromolecules such as enzymes. However, it is difficult to detect and purify the micromolecules such as small peptides, because of their instability and degradability. Construction of multi-copy recombinant expression plasmid can be achieved by inserting multiple target genes or expression cassette containing target genes with the same orientation into expression vector. This is effective to increase the expression level of small peptides. In this article we described four methods in order to provide some optional methods and ideas for the expression of active small peptides.
Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; metabolism ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4.3H2O, 0.062% MgSO4.7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4.7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662 +/- 0.041) g/L, which is about 13.7 folds of the one optimized before.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Liver ; chemistry ; Peptides ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sharks ; metabolism

Timely cell cycle regulation is conducted by sequential activation of a family of serine-threonine kinases called cycle dependent kinases (CDKs). Tight CDK regulation involves cyclin dependent kinase inhibitors (CKIs) which ensure the correct timing of CDK activation in different phases of the cell cycle. One CKI of importance is p27(KIP1). The regulation and cellular localization of p27(KIP1) can result in biologically contradicting roles when found in the nucleus or cytoplasm of both normal and tumor cells. The p27(KIP1) protein is mainly regulated by proteasomal degradation and its downregulation is often correlated with poor prognosis in several types of human cancers. The protein can also be functionally inactivated by cytoplasmic localization or by phosphorylation. The p27(KIP1) protein is an unconventional tumor suppressor because mutation of its gene is extremely rare in tumors, implying the normal function of the protein is deranged during tumor development. While the tumor suppressor function is mediated by p27(KIP1)'s inhibitory interactions with the cyclin/CDK complexes, its oncogenic function is cyclin/CDK independent, and in many cases correlates with cytoplasmic localization. Here we review the basic features and novel aspects of the p27(KIP1) protein, which displays genetically separable tumor suppressing and oncogenic functions.
Animals ; Cyclin-Dependent Kinases/genetics/*metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mutation ; Neoplasms/genetics/*metabolism ; Phosphorylation/genetics ; Protein Transport/genetics ; Tumor Suppressor Proteins/genetics/*metabolism

The cyanobacterial circadian clock has three relatively independent parts: the input path, the core oscillator, and the output path. The core oscillator is composed of three clock proteins: KaiA, KaiB, and KaiC. The interactions among these three proteins generate a rhythmic signal and convey the input signals to the output signals to maintain the accuracy and stability of the oscillation of downstream signals. Based on the cyanobacterial circadian clock and the structure, function, and interaction of the clock proteins of the core oscillator, combining the recent results from our laboratory, this review summarized the recent progresses of the molecular mechanism of KaiA in regulating KaiC's enzymatic activity, mediating phase reset of the oscillator, and competing with CikA for the binding site of KaiB.
Bacterial Proteins ; genetics ; metabolism ; Circadian Clocks ; genetics ; Circadian Rhythm Signaling Peptides and Proteins ; metabolism ; Cyanobacteria ; genetics ; Enzyme Activation ; genetics

AIMBy using of Escherichia coli DH5alpha to express GST-Ccd1 fusion protein and which was purified by affinity chromatographic separation. The purified target protein was used to immunize rabbits to prepare polyclonal antibody.

METHODSThe previously constructed recombinant prokaryotic expression vector pGEX-5X-1-Ccd1 was transformed into Escherichia coli DH5alpha and was induced to expression by IPTG. The recombinant target protein was expressed with soluble state in Escherichia coli expression system which was separated and purified by chromatographic column stuffed with glutathione Sepharose 4B. The prepared antigen was used to immunize rabbits to get anti-Ccd1 specific rabbit original polyclonal antibody.

RESULTSELISA data demonstrated that the antibody titer of the serum was up to 1:40 000. Immunohistochemistry analysis indicated that the "home-made" antibody had a specific interaction with Ccd1 protein and which could be used for extended experimental research.

CONCLUSIONThe anti-Ccd1 polyclonal antibody we had prepared had a high quality of potency and specificity. The antibody was demonstrated by experiments that which could totally fulfill the requirement of immunoblotting and immunohistochemistry study of Ccd1. The antibody provided an useful experimental tool to profoundly research the tissue expression profile, intercellular location and biological function of Ccd1.

Antibodies ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Glutathione Transferase ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification

OBJECTIVETo investigate the role of autophagy on the pathogenesis of spinocerebellar ataxia 3/Machado-Joseph disease (SCA3/MJD).

METHODSHEK293 cells expressing polyglutamine-expanded ataxin-3 were used as cell model for SCA3/MJD. The level of polyglutamine-expanded ataxin-3 was detected after cells were treated with different inhibitors or inducer of autophagy.

RESULTSInhibition of autophagy increased aggregate formation and cell death in HEK293 cells expressing mutated ataxin-3, and vice versa.

CONCLUSIONThe data suggested that autophagy is involved in the degradation of mutant ataxin-3, resulting in a decrease in the proportions of aggregate-containing cells and cell death in HEK293 cells expressing polyglutamine-expanded ataxin-3. It is possible that autophagy may be applied as a potential therapeutic approach for SCA3/MJD.

Ataxin-3 ; Autophagy ; Cell Line ; Humans ; Machado-Joseph Disease ; genetics ; metabolism ; physiopathology ; Mutation ; Nerve Tissue Proteins ; genetics ; metabolism ; Nuclear Proteins ; genetics ; metabolism ; Peptides ; metabolism ; Repressor Proteins ; genetics ; metabolism

Antimicrobial peptides (AMPs), a class of short proteins with a broad spectrum of antibacterial activities, are isolated from a wide variety of animals, both vertebrates and invertebrates, and plants as well as from bacteria and fungi. They are a key component of the innate immune response in most multicellular organisms. Owing to their potent, broad-spectrum antibacterial activities and uneasy developing of drug resistance, these peptides are of great clinical significance. However, preparation of AMPs at a large scale is a severe challenge to the development of the commercial products. Undoubtedly, construction of high-level biological expression systems for the production of AMPs is the key in its clinical application process. Herein, we summarize the progress in researches on heterogenous expression of AMPs in prokaryotic expression systems and eukaryotic expression systems.
Animals ; Antimicrobial Cationic Peptides ; biosynthesis ; chemistry ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Insecta ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Yeasts ; genetics ; metabolism

The flanking fragments of the whiE(a) gene cluster was PCR amplified, cloned and used to construct the gene replacement plasmid pHL643. pHL643 was conjugated into Streptomyces avermitilis NRRL8165 followed by screening for double crossover event, yielding three apramycin resistance and thiostrepton sensitive isolates named ZJ1, ZJ2 and ZJ3, which were deficient in biosynthesis of the grey spore pigment. The whiE(a) gene replacement of these isolates was confirmed by Southern hybridization. Fermentation of the mutant strains in shaking flasks and HPLC analyses showed that the production of avermectins increased by 47% compared with that of the wild type, indicating that the spore pigment biosynthesis competes with the avermectins biosynthetic pathway.
Bacterial Proteins ; biosynthesis ; genetics ; metabolism ; Cloning, Molecular ; Ivermectin ; analogs & derivatives ; metabolism ; Mutation ; Peptides ; genetics ; metabolism ; Spores, Bacterial ; genetics ; Streptomyces ; cytology ; genetics ; metabolism

Arabidopsis ; chemistry ; genetics ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Crystallography, X-Ray ; Intercellular Signaling Peptides and Proteins ; chemistry ; genetics ; metabolism ; Protein Conformation ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism

To study the expression of Dickkopf-1 (DKK-1) in endometrium of pregnant mice during the peri-implantation period and the role of DKK-1 during the embryo implantation in mice. Immunohistochemical technique was employed to determine the location of DKK-1 protein in endometrium, and semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was utilized to determine the levels of DKK-1 mRNA. Our results showed that the expressions of DKK1 mRNA and protein were higher in experimental groups than in control group (P<0.01) and it increased significantly on day 3 and reached its peak on day 4, and then decreased gradually on day 5-7. The levels of DKK-1 mRNA and protein on day 4 was significantly higher than those of other groups (P<0.01). It is concluded that DKK-1 probably plays an important role in signal transudation of embryo implantation and its high expression indicates the opening of implantation window.
Embryo Implantation/*genetics ; Endometrium/*metabolism ; Intercellular Signaling Peptides and Proteins/*biosynthesis ; Intercellular Signaling Peptides and Proteins/genetics ; Protein Biosynthesis ; RNA, Messenger/genetics ; Transcription, Genetic