Expression of acid cleavable Asp-Pro linked multimeric AFP peptide in E. coli

Background Difficult to express peptides are usually produced by co-expression with fusion partners. In this case, a significant mass part of the recombinant product falls on the subsequently removed fusion partner. On the other hand, multimerization of peptides is known to improve its proteolytic stability in E. coli due to the inclusion of body formation, which is sequence specific. Thereby, the peptide itself may serve as a fusion partner and one may produce more than one mole of the desired product per mole of fusion protein. This paper proposes a method for multimeric production of a human alpha-fetoprotein fragment with optimized multimer design and processing. This fragment may further find its application in the cytotoxic drug delivery field or as an inhibitor of endogenous alpha-fetoprotein. Results Multimerization of the extended alpha-fetoprotein receptor-binding peptide improved its stability in E. coli, and pentamer was found to be the largest stable with the highest expression level. As high as 10 aspartate-proline bonds used to separate peptide repeats were easily hydrolyzed in optimized formic acid-based conditions with 100% multimer conversion. The major product was represented by unaltered functional alpha-fetoprotein fragment while most side-products were its formyl-Pro, formyl-Tyr, and formyl-Lys derivatives. Single-step semi-preparative RP-HPLC was enough to separate unaltered peptide from the hydrolysis mixture. Conclusions A recombinant peptide derived from human alpha-fetoprotein can be produced via multimerization with subsequent formic acid hydrolysis and RP-HPLC purification. The reported procedure is characterized by the lower reagent cost in comparison with enzymatic hydrolysis of peptide fusions and solid-phase synthesis. This method may be adopted for different peptide expression, especially with low amino and hydroxy side chain content.

Synthesis of Biomaterial-Based Hydrogels Reinforced with Cellulose Nanocrystals for Biomedical Applications

Cellulose nanocrystals (CNC) were prepared by formic acid hydrolysis and TEMPO- (2,2,6,6-tetramethyl-piperidine-1-oxyl-) mediated oxidation. The prepared CNCs were reinforced into biopolymers chitosan (CHI), alginate (ALG), and gelatin (GEL) to obtain “CNC-ALG-GEL” and “CNC-CHI-GEL” hydrogels. The synthesized hydrogels were characterized for physicochemical, thermal, and structural characterization using Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), thermal gravity analysis (TGA), and X-ray diffraction (XRD) analyses. Notably, the reinforcement of CNC has not altered the molecular structure of a biopolymer as revealed by FT-IR analysis. The hydrogels reinforced with CNC have shown better thermal stability and miscibility as revealed by thermal gravity analysis. The physicochemical, thermal, and structural characterization revealed the chemical interaction and electrostatic attraction between the CNC and biopolymers. The biocompatibility was investigated by evaluating the viability of the L929 fibroblast cell, which represents good biocompatibility and nontoxic nature. These hydrogels could be implemented in therapeutic biomedical research and regenerative medicinal applications.

Composites polyvinyl alcohol filled with nanocellulose from oil palm waste by formic acid hydrolysis

Composites polyvinyl alcohol filled with nanocellulose from oil palm empty fruit bunch (OPEFB) and oil palm fronds (OPF) have investigated. Nanocellulose prepared by formic acid 50 wt%. Functional groups and degree of crystalline of nanocellulose were analyzed by FTIR and XRD. The degree of crystalline nanocellulose presented at 2θ at intensity of 22.26 and 21.86 for OPEFB and OPF. Amorphous part of nanocellulose at intensity of 14.68 and 15.86 for OPEFB and OPF. Functional group of nanocellulose give the same pattern for OPEFB and OPF. Nanocellulose reinforce in polyvinyl alcohol as many as 1, 5, 10 and 20% and as a comparison with polyvinyl alcohol without filler. Eight of composite films and one of vinyl alcohol film have studied mechanical properties such as tensile strength and elongation at break, and also functional groups. The tensile strength of OPF 5% and 10% increased than polyvinyl alcohol matrix. Tensile strength OPEFB 10% slightly increase from polyvinyl alcohol. The elongation of composites both OPEFB and OPF decreased with increased of nanocellulose loading.

Isolation and Characterization of Cellulose Nanofiber (CNF) from Sugarcane Bagasse by Acid Hydrolysis with Addition of Ferric Chloride Catalyst (FeCl3 )

Sugarcane bagass was used as effect of hydrolysis time for isolation of cellulose nanofiber (CNF) was done. The other components such as lignin and hemicellulose were removed from the biomass by adding NaOH and NaOCl and continue to synthesist of CNF has done using formic acid hydrolysis wiht addition of ferric chloride catalyst. FTIR analysis showed that were no significant variations in peak positions, This result did not affect the chemical compounds of CNF. XRD analysis showed increase the hydrolisis time also increase the crystallinity percentage and crystalline size. Increasing hydrolysis time would decreased the percentage yield of CNF.

Evaluation of Global Genomic DNA Methylation in Human Whole Blood by Capillary Electrophoresis UV Detection

Alterations in global DNA methylation are implicated in various pathophysiological processes. The development of simple and quick, yet robust, methods to assess DNA methylation is required to facilitate its measurement and interpretation in clinical practice. We describe a highly sensitive and reproducible capillary electrophoresis method with UV detection for the separation and detection of cytosine and methylcytosine, after formic acid hydrolysis of DNA extracted from human whole blood. Hydrolysed samples were dried and resuspended with water and directly injected into the capillary without sample derivatization procedures. The use of a run buffer containing 50 mmol/L BIS-TRIS propane (BTP) phosphate buffer at pH 3.25 and 60 mmol/L sodium acetate buffer at pH 3.60 (4 : 1, v/v) allowed full analyte identification within 11 min. Precision tests indicated an elevated reproducibility with an interassay CV of 1.98% when starting from 2 μg of the extracted DNA. The method was successfully tested by measuring the DNA methylation degree both in healthy volunteers and in reference calf thymus DNA.