Differential expression of human basic fibroblast growth factor in Escherichia coli: potential role of promoter

Four different expression systems were developed for expression of the cDNA encoding human basic fibroblast growth factor (hbFGF), using Escherichia coli as host organism. The hbfgf structural gene was cloned into four expression vectors, pET-3a, pTrc99A, pPR37 and pKK223-3 differing only in their promoters, which were T7, trc, PR and tac respectively. Expression of the gene was induced by adding 0.5 mM (final concentration) of isopropyl--D-thio-galactopyranoside (IPTG) for the vectors carrying T7, trc and tac promoters or by a temperature shift from 30 to 42 °C for the vector carrying PR. The highest level of expression was observed in pET-1005 (a pET-3a derivative)/BL21 (DE3) system with 18.5 mg/l rhbFGF and the second high level expression was in pR37-1007 (pPR37 derivative) BL21 (DE3) system with 5 mg of rhbFGF/l. Since in the latter system a temperature shift was used for induction, 29% of the hbFGF was recovered as inclusion bodies in the insoluble cell fraction. The level of expression for the two other systems (pTrc-1006 and pKK-1008) was very low.     

Flow injection amperometric detection of insulin at cobalt hydroxide nanoparticles modified carbon ceramic electrode

Cobalt hydroxide nanoparticles were prepared onto a carbon ceramic electrode (CHN|CCE) using the cyclic voltammetry (CV) technique. The modified electrode was characterized by X-ray diffraction and scanning electron microscopy. The results showed that CHN with a single-layer structure was uniformly electrodeposited on the surface of CCE. The electrocatalytic activity of the modified electrode toward the oxidation of insulin was studied by CV. CHN|CCE was also used in a homemade flow injection analysis system for insulin determination. The limit of detection (signal/noise [S/N] = 3) and sensitivity were found to be 0.11 nM and 11.8 nA/nM, respectively. Moreover, the sensor was used for detection of insulin in human serum samples. This sensor showed attractive properties such as high stability, reproducibility, and high selectivity.     

The effects of somatic mutations on EGFR interaction with anti-EGFR monoclonal antibodies: Implication for acquired resistance

A number of mutations in the epidermal growth factor receptor (EGFR) have been identified that imparts resistance to anti-EGFR monoclonal antibodies (mAbs) in clinical and preclinical samples. Primary or acquired resistance to targeted therapy will eventually limit the clinical benefit of anticancer mAbs. The aim of the current study was to perform computational analysis to investigate the structural implications of the EGFR somatic mutations on its complexes with the four anti-EGFR mAbs (Cetuximab, Panitumumab, Necitumumab, and Matuzumab). Docking analysis and molecular dynamics (MD) simulations were performed to understand the plausible structural and dynamical implications caused by somatic mutations available in the Catalogue of Somatic Mutations in Cancer database on the EGFR and anti-EGFR mAbs. We found that EGFR^S492R and EGFR^V441I in complex with Cetuximab, EGFR^R377S and EGFR^S447Y in complex with Panitumumab, and EGFR^V441I in complex with Necitumumab have a weakest binding affinity in comparison to EGFR^WT in complex with the relevant mAb. Taken together with the results obtained from docking analysis and MD simulations, the present findings may suggest that, the S492R and V441I mutations confer resistance to Cetuximab, R377S and S447Y mutations mediate resistance to Panitumumab and finally, V441I mutation also confers resistance to Necitumumab.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Proline dehydrogenase from Pseudomonas fluorescens: Gene cloning, purification, characterization and homology modeling

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescens was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K _m and V _max values of the P. fluorescens ProDH were estimated to be 35 mM and 116 μmol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30°C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescens ProDH.     

Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84

The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K _m and V _max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.     

Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems

This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.     

Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase

This work presents a comprehensive study between the polymer/salt aqueous two-phase systems (ATPS) and chromatography process for downstream processing of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH). First, the partitioning behavior of recombinant PheDH in polyethylene glycol (PEG)/K₂HPO₄ ATPS was examined. For comparative purpose, a classical chromatographic protocol was performed as well. Investigation of chromatography and ATPS procedures revealed that the ATPS comprising of 9% (w/w) PEG-6000, 16% (w/w) K₂HPO₄ and 16% (w/w) KCl with pH of 8.0, volume ratio (V _R ) of 0.25, temperature of 25 °C and 40% (w/w) cell lysate ensured the most favorable approach for PheDH downstream process. A specific activity of 4,231.4 U/mg, a yield of 96.7% and a recovery of 162.0% were obtained. Furthermore, the shorter process time (4 vs. 48 h) and the lower total cost (4 vs. 20 €) were additionally features that confirmed the suitability of proposed technique.     

Cloning and expression of Bacillus sphaericus phenylalanine dehydrogenase gene in Bacillus subtilis cells: purification and enzyme properties

Cloning and expression of the L-phenylalanine dehydrogenase (PheDH) gene from Bacillus sphaericus in B. subtilis was performed. It was ligated into the pHY300PLK shuttle vector and the resulting plasmid, pHYDH encoding polypeptide with molecular weight of 340 kDa, then transformed in B. subtilis ISW1214 and Escherichia coli JM109 competent cells for expression. Bacillus subtilis ISW1214/pHYDH only produced PheDH enzyme (4700 U/l). The recombinant PheDH was purified to near homogeneity as judged by SDS–polyacrylamide gel electrophoresis (M _r 41000 Da) and the result was 40-fold with a yield of about 54%. Apparent K _m values for L-phenylalanine (Phe), L-tyrosine and NAD⁺ were 0.24, 0.48 and 0.19 mM respectively. The optimum pH of the recombinant enzyme was 11 for the oxidative deamination, 10.2 for the reductive amination. The features of recombinant PheDH enzyme were comparable with the wild type PheDH protein.