Using sno-lncRNAs as potential markers for Prader-Willi syndrome diagnosis.

The genetic disorder Prader-Willi syndrome (PWS) is mainly caused by the loss of multiple paternally expressed genes in chromosome 15q11-q13 (the PWS region). Early diagnosis of PWS is essential for timely treatment, leading to effectively easing some clinical symptoms. Molecular approaches for PWS diagnosis at the DNA level are available, but the diagnosis of PWS at the RNA level has been limited. Here, we show that a cluster of paternally transcribed snoRNA-ended long noncoding RNAs (sno-lncRNAs, sno-lncRNA1-5) derived from the SNORD116 locus in the PWS region can serve as diagnostic markers. In particular, quantification analysis has revealed that 6,000 copies of sno-lncRNA3 are present in 1 µL whole blood samples from non-PWS individuals. sno-lncRNA3 is absent in all examined whole blood samples of 8 PWS individuals compared to 42 non-PWS individuals and dried blood samples of 35 PWS individuals compared to 24 non-PWS individuals. Further developing a new CRISPR-MhdCas13c system for RNA detection with a sensitivity of 10 molecules per µL has ensured sno-lncRNA3 detection in non-PWS, but not PWS individuals. Together, we suggest that the absence of sno-lncRNA3 represents a potential marker for PWS diagnosis that can be detected by both RT-qPCR and CRISPR-MhdCas13c systems with only microlitre amount of blood samples. Such an RNA-based sensitive and convenient approach may facilitate the early detection of PWS.

[Expression and purification of recombinant human interleukin 4 in Escherichia coli].

Human interleukin 4 (IL-4) cDNA was optimized and synthesized according to E. coli preferred codon. A recombinant expression plasmid pET-30a (+)/rhIL-4 was constructed with the target cDNA inserted between Nde I and EcoR I sites, which can translate the mature IL-4 protein with an extra methionine residue at N-terminal. The expression vector was transformed into E. coli BL21 (DE3). The rhIL-4 protein was expressed in the inclusion body. By using the optimized fermentation conditions, the high expression level was achieved with the expression level as high as 35% of total protein obtained. A purification strategy has been designed which includes Q-Sepharose and SP-Sepharose ion-exchange chromatography and dialysis renaturation. The rhIL-4 was purified with the purity more than 98% and the yield of 40 mg per liter fermentation culture achieved. Western blot proved that the purified protein is IL-4. Amino acid sequencing revealed that N-terminal 16 residue sequence is identical to the theoretical sequence. Biological activity assay on TF-1 cells demonstrated that the rhIL-4 is active with an activity of 2.5 x 10(6) AU/mg. This study promises large scale production of rhIL-4.

Human proinsulin C-peptide from a precursor overexpressed in Pichia pastoris.

In this article we report the production of human proinsulin C-peptide with 31 amino acid residues from a precursor overexpressed in Pichia pastoris. A C-peptide precursor expression plasmid containing nine C-peptide genes in tandem was constructed and used to transform P. pastoris. Transformants with a high copy number of the C-peptide precursor gene integrated into the chromosome of P. pastoris were selected. In high-density fermentation in a 300 liter fermentor using a simple culture medium composed mainly of salt and methanol, the C-peptide precursor was overexpressed to a level of 2.28 g per liter. A simple procedure was established to purify the expression product from the culture medium. The purified C-peptide precursor was converted into C-peptide by trypsin and carboxypeptidase B joint digestion. The yield of C-peptide with a purity of 96% was 730 mg per liter of culture. The purified C-peptide was characterized by mass spectrometry, N- and C-terminal amino acid sequencing, and sodium dodecylsulfate-polyacrylamide gel electrophoresis.