Phenylketonuria (PKU) is an inherited autosomal recessive disorder of phenylalanine metabolism, mainly caused by a deficiency of phenylalanine hydroxylase (PAH). The incidence of various PAH mutations differs among race and ethnicity. Here we report a spectrum of PAH mutations complied from 796 PKU patients from mainland China. The all 13 exons and adjacent intronic regions of the PAH gene were determined by next-generation sequencing. We identified 194 different mutations, of which 41 are not reported before. Several mutations reoccurred with high frequency including p.R243Q, p.EX6-96A > G, p.V399V, p.R241C, p.R111*, p.Y356*, p.R413P, and IVS4-1G > A. 76.33% of mutations were localized in exons 3, 6, 7, 11, 12. We further compared the frequency of each mutation between populations in northern and southern China, and found significant differences in 19 mutations. Furthermore, we identified 101 mutations that are not reported before in Chinese population, our study thus broadens the mutational spectrum of Chinese PKU patients. Additionally, 41 novel mutations will expand and improve PAH mutation database. Finally, our study offers proof that NGS is effective, reduces screening times and costs, and facilitates the provision of appropriate genetic counseling for PKU patients.

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We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or pT7His (His 10 -mDsRed) prokaryotic expression vectors. Then the fluorescent proteins were expressed in Rosetta (DE3) pLysS by IPTG induction or autoinduction. We purified the fluorescent proteins by affinity chromatography (Amylose and metal ionchelating column), anion-exchange chromatography (High Q column), size exclusive chromatography (Sephacryl S-200 column), and hydrophobic interaction chromatography (Methyl HIC column) to exhibit the protein-purification techniques. After purification, the fusion protein MBP-EGFP was cleaved by TEV protease. The recombinant mDsRed protein was crystallized by hanging drop vapor diffusion technique to show students the basic operation of crystallization. The whole procedure can be monitored real time by naked eyes when using fluorescent proteins. The demonstration of expression, purification, crystallization, and protease cleavage became much more vivid and interesting, which greatly deepened the students' understanding of modern protein-science techniques.Keywords: Green fluorescent protein, red fluorescent protein DsRed monomer, chromatography purification, protein crystallization, TEV protease cleavage.We have introduced a course named ''modern techniques in protein structural and functional analysis'' to the senior undergraduate students or the first-year graduate students who studied biology but had not yet taken similar courses. The course focused on the whole process of cloning, expression, purification, functional characterization and structural analysis of a specific protein. Green and red fluorescent proteins (GFP and RFP) were used in the laboratory sections, which greatly increased the students' interests and minimized the specialized equipment usage at the same time.The GFP, a small protein comprised 238 amino acids from the jellyfish Aequorea victoria, emits green fluorescent light when exposed to blue light [1]. It is stable under most physiological environment as a weak dimer [2]. Nowadays, GFP has already become an excellent marker for gene expression and protein localization in cellular and molecular biology. It is also used to demonstrate the dynamic processes of many biological activities within the cell, including protein folding, and transport. The GFP gene can also be maintained through breeding, and it was used to develop versatile reporter systems for genetic analysis.Different mutants of GFP have been engineered over the last few years. The enhanced green fluorescent protein (EGFP) 1 was one of the most famous mutants with increased stability and fluorescence efficiency. EGFP shifts its major excitation peak from 395 to 488 nm while the peak emission remains at 507 nm [3]. Using commercial vectors such...

The crystal structures of almost all the enzymes of arginine metabolism have been determined, but arginine decarboxylase's structure is not resolved yet. In order to characterize and crystallize arginine decarboxylase, we overexpressed biosynthetic arginine decarboxylase (ADC; EC 4.1.1.19, encoded by the speA gene) from Escherichia coli in the T7 expression system as a cleavable poly-His-tagged fusion construct. The expressed recombinant His(10)-ADC (77.3 kDa) was first purified by Ni-NTA affinity chromatography, then proteolytically digested with Tobacco Etch Virus (TEV) protease to remove the poly-His fusion tag, and finally purified by anion exchange chromatography. The His(10) tag removed recombinant ADC (74.1 kDa)'s typical yield was 90 mg from 1 l of culture medium with purity above 98%. The recombinant ADC was assayed for decarboxylase activity, showing decarboxylase activity of 2.8 U/mg, similar to the purified native E. coli ADC. The decarboxylase activity assay also showed that the purified recombinant ADC tolerated broad ranges of pH (pH 6-9) and temperature (20-80 degrees C). Our research may facilitate further studies of ADC structure and function, including the determination of its crystal structure by X-ray diffraction.