Lixin Ma

Toward an understanding of the protein interaction network of the human liver.

Proteome‐scale protein interaction maps are available for many organisms, ranging from bacteria, yeast, worms and flies to humans. These maps provide substantial new insights into systems biology, disease research and drug discovery. However, only a small fraction of the total number of human protein–protein interactions has been identified. In this study, we map the interactions of an unbiased selection of 5026 human liver expression proteins by yeast two‐hybrid technology and establish a human liver protein interaction network (HLPN) composed of 3484 interactions among 2582 proteins. The data set has a validation rate of over 72% as determined by three independent biochemical or cellular assays. The network includes metabolic enzymes and liver‐specific, liver‐phenotype and liver‐disease proteins that are individually critical for the maintenance of liver functions. The liver enriched proteins had significantly different topological properties and increased our understanding of the functional relationships among proteins in a liver‐specific manner. Our data represent the first comprehensive description of a HLPN, which could be a valuable tool for understanding the functioning of the protein interaction network of the human liver.

Subunit-subunit interactions in the human 26S proteasome.

Ubiquitin‐dependent proteolysis is mediated by the proteasome. To understand the structure and function of the human 26S proteasome, we cloned complete ORFs of 32 human proteasome subunits and conducted a yeast two‐hybrid analysis of their interactions with each other. We observed that there are 114 interacting‐pairs in the human 26S proteasome. About 10% (11/114) of these interacting‐pairs was confirmed by the GST‐pull down analysis. Among these observed interacting subunits, 58% (66/114) are novel and the rest 42% (48/114) has been reported previously in human or in other species. We observed new interactions between the 19S regulatory particle and the β‐rings of the 20S catalytic particle and therefore proposed a modified model of the 26S proteasome.

High-throughput cloning of human liver complete open reading frames using homologous recombination in Escherichia coli

In this article, we describe a high-throughput cloning method, seamless enzyme-free cloning (SEFC), which allows one-step assembly of DNA fragments in vivo via homologous recombination in Escherichia coli. In the method, the desired open reading frame (ORF) is amplified by use of ORF-specific primers with flanking sequences identical to the two ends of a linearized vector. The polymerase chain reaction (PCR) product and the linearized vector are then cotransformed into E. coli cells, where the ORF is incorporated into the vector in vivo. SEFC is a simple, reliable, and inexpensive method of cloning in which PCR fragments are fused into expression vectors without unwanted amino acids or extra in vitro manipulations apart from the single PCR amplification step. Using this method, we successfully cloned human liver complete ORFs into the yeast AD and DB vectors and generated a clone resource of 4964 AD-ORFs and 4676 DB-ORFs in 3months. This approach will be useful for daily DNA cloning and for creating proteome-scale clone resources.

Recombinant expression of chitosanase from Bacillus subtilis HD145 in Pichia pastoris

A chitosanase-producing bacterium, Bacillus subtilis HD145 CCTCC AB 2010353, was isolated from a soil sample. The gene (csn) encoding chitosanase was cloned, sequenced, and expressed in the Pichia pastoris strain as a soluble and active form. Its expression level could be as high as 800 mg/L, and enzymatic activity reached approximately 9000 U/mg. The optimum pH and temperature were 5.5 and 50 °C, respectively. The recombinant protein was partially glycosylated. Its half lives at temperatures of 50 and 60 °C were 26 h and 23 min, respectively. Enzymatic activity was increased with an increasing degree of deacetylation of chitosan. The enzymatic productions of chitooligosaccharides from chitosans of various deacetylation degrees mainly ranged from chitobiose to chitopentamer.

Anti-tumor activity of metformin: from metabolic and epigenetic perspectives.

Metformin has been used to treat type 2 diabetes for over 50 years. Epidemiological, preclinical and clinical studies suggest that metformin treatment reduces cancer incidence in diabetes patients. Due to its potential as an anti-cancer agent and its low cost, metformin has gained intense research interest. Its traditional anti-cancer mechanisms involve both indirect and direct insulin-dependent pathways. Here, we discussed the anti-tumor mechanism of metformin from the aspects of cell metabolism and epigenetic modifications. The effects of metformin on anti-cancer immunity and apoptosis were also described. Understanding these mechanisms will shed lights on application of metformin in clinical trials and development of anti-cancer therapy.

High-level expression of endo-β-N-acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and its application for the deglycosylation of glycoproteins.

Endo-β-N-acetylglucosaminidase H (Endo H, EC3.2.1.96) is a glycohydrolase that is widely used in the study of glycoproteins. The present study aimed to assess the effect of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris. The DNA coding sequence of this enzyme was optimized based on the codon usage bias of Pichia pastoris and synthesized through overlapping PCR. This novel gene was cloned into a pHBM905A vector and introduced into Pichia pastoris GS115 for secretary expression. The yield of the target protein reached approximately 397 mg/l after a 6-d induction with 1% (v/v) methanol in shake flasks, which is much higher than that observed upon heterologous expression in Escherichia coli and silkworm. This recombinant enzyme was purified and its enzymatic features were studied. Its specific activity was 461573 U/mg. Its optimum pH and temperature were pH 5.5 and 37°C, respectively. Moreover, our study showed that the N-linked glycan side-chains of several recombinant proteins expressed in Pichia pastoris can be efficiently removed through either the co-fermentation of this recombinant strain with strains expressing substrates or by mixing the cell culture supernatants of the endo-β-N-acetylglucosaminidase H expressing strain with strains expressing substrates after fermentation. This is the first report of high-level endo-β-N-acetylglucosaminidase H expression in Pichia pastoris and the application of this enzyme in the deglycosylation of raw glycoproteins heterologously expressed in Pichia pastoris using simplified methods.

An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases

Abstract Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.

Expression, activation and characterization of porcine trypsin in Pichia pastoris GS115.

Trypsin is a typical member of serine protease families, specifically cleaving the carboxyl group of peptides at the basic amino acids arginine and lysine. The gene fragment of porcine trypsin with its propeptide coding sequence was optimized and synthesized according to the codon usage bias of Pichia pastoris. The optimized sequence was integrated into the genome of P. pastoris GS115 using the vector pHBM905A. The yield of the recombinant protein was 0.48mg/ml with a maximum activity of 19.2U/ml after 96-h induction in a 5-l fermenter. An optimum activity for the recombinant trypsin was observed at 35°C and pH 8.5. This is the first time to express the porcine trypsinogen with P. pastoris expression system. This report also found that the propeptide was cleaved from the recombinant protein and the enzymogen was transferred into trypsin at the later phase of the fed-batch cultivation. In particular, the activation process can be initiated by changing pH.

Improved method for constructing plant amiRNA vectors with blue–white screening and MAGIC

Artificial microRNA (amiRNA) technology is a novel tool in reverse genetic research for discovering or validating gene functions in plants. A convenient cloning strategy has been developed to construct plant amiRNA vectors based on lacO reconstruction and mating-assisted, genetically-integrated cloning (MAGIC). The amiRNA precursor fragment was generated by PCR and inserted into a small donor plasmid through reconstruction of integrated lacO sequence. Blue recombinants were selected on plates containing X-gal and the efficiency of successful clones was 100%. The amiRNA expression cassette was transferred from the donor plasmid to the recipient plasmid p1301-gfp through MAGIC and an amiRNA expression plasmid was created. More than 40 plant amiRNA vectors were generated through this method, one of which was transformed into Arabidopsis thaliana and the target gene was silenced efficiently. The approach will be useful for amiRNA expression vectors construction in plants.

Efficient vectors for expression cloning of large numbers of PCR fragments in P. pastoris.

The yeast vectors described, pYEV and pYEVB, were designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) and immediate protein expression in Pichia pastoris. The pYEV vector was used to clone PCR fragments obtained by using Taq or similar polymerase mixes, which leave an A‐base overhang. The other vector pYEVB, with the same features for blunt‐end ligation of PCR products, was developed to be complementary to pYEV. These two plasmids were linearized using the restriction enzymes BfuI and SchI, respectively. The purified PCR products, without any other treatments, were cloned into the linearized vectors, followed by selection on plates supplemented with X‐gal. Only desired recombinants carrying the target gene in the correct orientation can give typical blue colonies. This screening technique is based on a lacO reconstruction strategy that can produce a full‐length lacO to exhaust endogenous LacI and switch on the transcription of lacZ in the host. The recombinant plasmids extracted from the blue colonies can be linearized by SalI and transformed into P. pastoris for immediate expression. By using these two vectors, researchers could be saved from the tedious and time‐consuming conventional cloning procedures. Copyright