Recombinant expression, purification and antimicrobial activity of a novel antimicrobial peptide PaDef in Pichia pastoris.

The antimicrobial peptide PaDef was isolated from Mexican avocado fruit and was reported to inhibit the growth of Escherichia coli and Staphylococcus aureus in 2013. In this study, an N-terminal 6 × His tagged recombinant PaDef (rPaDef) with a molecular weight of 7.5 KDa, for the first time, was expressed as a secreted peptide in Pichia pastoris. The optimal culture condition for rPaDef expression was determined to be incubation with 1.5% methanol for 72 h at 28 °C under pH 6.0. Under this condition, the amount of the rPaDef accumulation reached as high as 79.6 µg per 1 ml of culture medium. Once the rPaDef peptide was purified to reach a 95.7% purity using one-step nickel affinity chromatography, its strong and concentration-dependent antimicrobial activity was detected to be against a broad-spectrum of bacteria of both Gram-negative and Gram-positive. The growth of these bacterial pathogens was almost completely inhibited when the rPaDef peptide was at a concentration of as low as 90 µg/ml. In summary, our data showed that rPaDef derived from Mexican avocado fruit can be expressed and secreted efficiently when P. pastoris was used as a cell factory. This is the first report on heterologous expression of PaDef in P. pastoris and the approach described holds great promise for antibacterial drug development.

Expression, purification and initial characterization of a novel recombinant antimicrobial peptide Mytichitin-A in Pichia pastoris.

Mytichitin-A is an antimicrobial peptide isolated from the serum of Mytilus coruscus and is reported to inhibit bacterial growth as tested on several Gram-positive bacteria. To produce large quantity of Mytichitin-A to further investigate its biological activity, nucleotide sequence encoding a recombinant 6 × His-Mytichitin-A (rMytichitin-A) peptide was synthesized and inserted into the inducible yeast expression vector pPICZαA. With the availability of such an expression vector called pPICZαA-Mytichitin-A, we transformed Pichia pastoris GS115 cells with a SacI-linearized pPICZαA-Mytichitin-A by electroporation. Transgenic strains secreting rMytichitin-A with a molecular weight of approximate 10 KDa as expected were obtained. The optimal culture condition for rMytichitin-A expression was determined to be 1.0% methanol induction, 96 h incubation at 28 °C and the amount of rMytichitin-A reached 45.5 µg/ml. The percentage of rMytichitin-A was estimated to be 73.6% of the total protein. After rMytichitin-A was purified using nickel ions affinity chromatography, approximate 9.1 mg pure rMytichitin-A was obtained from 500 ml of cell culture medium with 97.8% purity. More importantly, both the culture supernatant and purified rMytichitin-A inhibited the growth of Gram-positive bacteria, especially Staphylococcus aureus and Bacillus subtilis with a minimum inhibition concentration of as low as 31 and 48 µg/ml, respectively. Differently from the native protein, however, the rMytichitin-A is not active against Gram-negative bacteria. Taken together, this is the first report on the heterologous expression of Mytichitin-A in P. pastoris. Our study showed that P. pastoris is an effective expression system for producing large quantities of biologically active Mytichitin-A for both research and application purposes.

Psychometric properties of a mainland Chinese version of the Iowa Infant Feeding Attitude Scale among postpartum women in China.

BACKGROUND: The Iowa Infant Feeding Attitude Scale (IIFAS) is used to evaluate maternal infant feeding attitude. The breastfeeding rate has declined but no validated instruments to assess infant feeding attitudes or related studies have been available in mainland China. AIMS: The purpose of this study was to assess the reliability and validity of a mainland Chinese version of the IIFAS among postpartum women. METHODS: Postpartum women (N = 660) were recruited from three university hospitals in Guangzhou in mainland China. The participants completed an IIFAS questionnaire before being discharged and accepted telephone follow-up sessions at 6 weeks and 3 months postpartum. The reliability of the scale was evaluated using intra-class correlations (ICC) for test-retest reliability, correlated item-total correlations and Cronbach's a. The validity of the scale was evaluated using the content validity index (CVI), construct validity using exploratory factor analysis and predictive validity using independent t-tests. RESULTS: The ICC was 0.861. The correlated item-total correlations ranged from 0.262-0.691. Cronbach's a was 0.623. The CVI was 0.996. Four factors were extracted using exploratory factor analysis and they contributed to 48.69% of the total variance. CONCLUSIONS: The mainland Chinese version of the IIFAS scale can be considered a reliable, valid and predictive scale for assessing infant feeding attitudes among women in mainland China. In-hospital scores on the scale were significant predictors of the infant feeding method and breastfeeding duration at 3 days, 6 weeks and 3 months postpartum. Construct validity was confirmed and showed four factors. However, future studies are required to improve the lower level internal consistency of the IIFAS.

Michael addition of pyrimidine with disaccharide acrylates catalyzed in organic medium with lipase M from Mucor javanicus.

The Michael addition of fluorouracil (0.01 mol), uracil (0.01 mol), thymine (0.01 mol) to disaccharide acrylates (0.03 mol) was carried out in pyridine (20 ml) at 50 degrees C for 72 h with lipase M from Mucor javanicus (200 mg). Six new pyrimidine derivatives containing a branched sugar were obtained in yields from 56 to 75%.