Gang Jia

Role of Akirin in Skeletal Myogenesis

Akirin is a recently discovered nuclear factor that plays an important role in innate immune responses. Beyond its role in innate immune responses, Akirin has recently been shown to play an important role in skeletal myogenesis. In this article, we will briefly review the structure and tissue distribution of Akirin and discuss recent advances in our understanding of its role and signal pathway in skeletal myogenesis.

Porcine phosphotyrosine interaction domain containing 1 modulates 3T3-L1 preadipocyte proliferation and differentiation

The phosphotyrosine interaction domain containing 1 (PID1) gene was firstly isolated from obese subjects and involved in obesity-associated insulin resistance. In the present study, Duroc×Landrace×Yorkshire (DLY) pig PID1 cDNA was cloned. The entire open reading frame of the cloned porcine PID1 is 654 bp. The predicted protein is composed of 217 amino acids residues with a molecular mass of 24,774 Da. Over-expression of porcine PID1 significantly accelerated the proliferation of 3T3-L1 preadipocyte, but inhibited preadipocyte differentiation by decreasing the numerous fat droplets appeared and down-regulating the mRNA expression levels of peroxisome proliferators-activated receptor-γ, CCAAT/enhancer binding protein α, fat acid synthase and lipoprotein lipase. Together, these results suggest that porcine PID1 plays a role in regulating adipose development.

Partial Optimization of the 5-Terminal Codon Increased a Recombination Porcine Pancreatic Lipase (opPPL) Expression in Pichia pastoris

Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P.pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), Km (0.041 mM), and Vmax (2.008 µmol.mg protein −1.min−1) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg.L−1 vs 36 mg.L−1) and total enzyme activity increased about 5 folds (1900 IU.L−1 vs 367 IU.L−1) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.

Role of NYGGF4 in insulin resistance

Insulin resistance is a clinical condition that is characterized by reducing glucose uptake in response to insulin. A major factor in the development of insulin resistance syndrome is obesity. NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese subjects. NYGGF4 induced the secretion of FFAs and TNF-α and caused mitochondrial dysfunction, which may cause insulin resistance. This review will summarize the effect of NYGGF4 on the adipogenesis, glucose uptake and mitochondrial dysfunction in vitro, and the possible mechanism and signal pathway of NYGGF4 for insulin resistance.

Effect of Porcine Akirin2 on Skeletal Myosin Heavy Chain Isoform Expression

Akirin2 plays an important role in skeletal myogenesis. In this study, we found that porcine Akirin2 (pAkirin2) mRNA level was significantly higher in fast extensor digitorum longus (EDL) and longissimus lumborum (LL) muscles than in slow soleus (SOL) muscle of pigs. Overexpression of pAkirin2 increased the number of myosin heavy chain (MHC)-positive cells, indicating that pAkirin2 promoted myoblast differentiation. We also found that overexpression of pAkirin2 increased the mRNA expressions of MHCI and MHCIIa and decreased the mRNA expression of MHCIIb. Myocyte enhancer factor 2 (MEF2) and nuclear factor of activated T cells (NFAT) are the major downstream effectors of calcineurin. Here we also observed that the mRNA expressions of MEF2C and NFATc1 were notably elevated by pAkirin2 overexpression. Together, our data indicate that the role of pAkirin2 in modulating MHCI and MHCIIa expressions may be achieved through calcineurin/NFATc1 signaling pathway.

Selenoprotein Genes Exhibit Differential Expression Patterns Between Hepatoma HepG2 and Normal Hepatocytes LO2 Cell Lines.

The purpose of this study was to compare messenger RNA (mRNA) expression of selenoprotein genes between hepatoma HepG2 and normal hepatocytes LO2 cell lines. Liver HepG2 and LO2 cells were cultured in 12-well plates under the same condition until cells grew to complete confluence, and then cells were harvested for total RNA and protein extraction. The qPCRs were performed to compare gene expression of 14 selenoprotein genes and 5 cancer signaling-related genes. Enzyme activities were also assayed. The results showed that human hepatoma HepG2 cells grew faster than normal hepatocytes LO2 cells. Among the genes investigated, 10 selenoprotein genes (Gpx1, Gpx3, Gpx4, Selx, Sepp, Sepw1, Sepn1, Selt, Seli, Selh) and 3 cancer signaling-related genes (Bcl-2A, caspase-3, and P38) were upregulated (Pu2009<u20090.05), while Selo and Bcl-2B were downregulated (Pu2009<u20090.05) in hepatoma HepG2 cells compared to LO2 cells. Significant correlations were found between selenoprotein genes and the cancer signaling-related genes Caspase3, P53, Bc1-2A, and Bc1-2B. Our results revealed that selenoprotein genes were aberrantly expressed in hepatoma HepG2 cells compared to normal liver LO2 cells, which indicated that those selenoprotein genes may play important roles in the occurrence and development of liver carcinogenesis.

STEAP4 and insulin resistance

Obesity is a multifactorial disease that caused by the interactions between genetic susceptibility genes and environmental cues. Obesity is considered as a major risk factor of insulin resistance. STEAP4 is a novel anti-obesity gene that is significantly down-regulated in adipose tissue of obese patients. Over-expression of STEAP4 can improve glucose uptake and mitochondrial function, and increase insulin sensitivity. STEAP4 expression is regulated by a variety of inflammatory cytokines, hormones, or adipokines. In this review, we discuss function of STEAP4 in regulating insulin resistance in adipose tissue in vivo, as well as in adipocytes in vitro.

Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system.

Parasin I (PI) is a 19 amino acid peptide with potent antimicrobial activities against a broad spectrum of microorganisms and is a good candidate for development as a novel antimicrobial agent. The objective of this study was to express and characterize a codon optimized parasin I peptide fused with human lysozyme (hLY). A 513 bp cDNA fragment encoding the mature hLY protein and parasin I peptide was designed and synthesized according to the codon bias of Pichia pastoris. A 4×Gly flexible amino acid linker with an enterokinase cleavage (DDDDK) was designed to link the PI to the C-terminal of hLY. The codon optimized recombinant hLY-PI was cloned into the pPICZαA vector and expressed in P. pastoris. The over-expressed extracellular rehLY-PI was purified using Ni sepharose affinity column and exhibited a molecular mass of approximately 18 kDa. After digested with enterokinase the rehLY-PI protein release its corresponding rehLY and rePI, with molecular mass of 16 kDa and 2 kDa, respectively, on Tricine-SDS-PAGE. The released rehLY exhibited similar lytical activity against Micrococcus lysodeikticus to its commercial hLY. The digested rehLY-PI product exhibited antimicrobial activities against Bacillus subtilis, Staphylococcus aureus and Escherichia coli, and synergism has been found between the released rePI and rehLY. In conclusion, we successfully optimized a rehLY-PI fusion protein encoding gene and over-expressed the rehLY-PI in P. pastoris. The recombination protein digested with enterokinase released functional hLY and antimicrobial parasin I, which demonstrates a potential for future use as an animal feed additive to partly replace antibiotic.

Codon optimization of Aspergillus niger feruloyl esterase and its expression in Pichia pastoris

Abstract To enhance the expression level of recombinant feruloyl esterase in Pichia pastoris, the Aspergillus niger feruloyl esterase (AnFaeA) gene was optimized based on the codon usage bias of P. pastoris and synthesized. The synthetic AnFaeA-syn was inserted into the pPICZαA and then integrated into P. pastoris X33. SDS-PAGE analysis of recombinant feruloyl esterase displayed a single protein band with an apparent molecular mass of 40.0 kDa. Western blot analysis confirmed that the expressed protein was the target protein. Furthermore, we showed that codon optimization increased enzyme yield for about 6.4-fold (35.12 U/mL vs 5.51 U/mL) in shake flask cultivation, compared to the non-codon-optimized control. Taken together, this study showed that the yield of feruloyl esterase could be increased by codon optimization.

Prokaryotic expression and characterization of a keratinolytic protease from Aspergillus niger

Abstract In this study, a keratinolytic protease gene (named as kerD) from Aspergillus niger was cloned. The full-length coding sequence of kerD consists of 1,251 bp and encodes a protein with 416 amino acid residues with a molecular mass of 43,831 Da. A DNA fragment encoding mature kerD without its signal sequence was inserted into the expression vector pET30a(+) and successfully expressed in Escherichia coli. The recombinant protein was purified to approximately 100% purity using Ni-IDA affinity chromatography, and identified by Western blot. The recombinant enzyme had an optimal pH of 8.0 and was stable at pH 7.0-9.0. It exhibited an optimal temperature for activity of 70 °C and was stable at 30-50 °C. It was highly inhibited by 1,10-phenanthroline, ethylenediaminetetraacetic acid and sodium dodecyl sulphate, but activated by phenylmethanesulfonyl fluoride, Mg2+, Fe2+, Mn2+, Cu2+, Zn2+, Ca2+, dithiothreitol, Triton X-100, dimethyl sulfoxide and isopropyl alcohol. The recombinant enzyme could hydrolyse a broad range of protein substrates.