Affinity purification and characterization of recombinant Bacillus sphaericus phenylalanine dehydrogenase produced by pET expression vector system.

Cloning and expression of the L-phenylalanine dehydrogenase gene, from B. sphaericus in E. coli were done. The gene was cloned in the vector pET16b and transformed into E. coli BL21 [DE3]. The functional form of the L-phenylalanine dehydrogenase enzyme was purified by affinity purification techniques, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Blue 4. Approximately 3 mg of highly purified recombinant enzyme was obtained from 950 mg cell pellet [wet weight]. The Relative molecular mass of the L-phenylalanine subunits was about 41 kDa by 10% SDS-PAGE. Using this method, the enzyme was obtained with a yield of 28%, and had a specific activity of 577.3 U/mg proteins, which is purified 88 times. This method was provided a facile and effective way for preparing the enzyme with a good yield that suitable for analytical purposes

Apolipoprotein E polymorphism in an Iranian hypercholesterolemic population

Apolipoprotein E [apo E] is a structural constituent of several serum lipoprotein classes. It plays an important role in lipid metabolism by acting as a ligand for low-density lipoprotein [LDL] and chylomicron remnant receptors. Three common alleles called epsilon 2, epsilon 3 and 4 have been described, which code for three protein isoforms [E2, E3 and E4]. The polymorphism is clinically significant, and it has therefore become very important in epidemiological studies attempting to obtain complete information about apo E allele frequencies in populations around the world. In the present study, two point mutations coding amino acid residues 112 and 158 were amplified using the polymerase chain reaction [PCR] from DNA extracted from 100 Iranian subjects. Apo E genotypes were determined by restriction fragment length polymorphism [RFLP] and allelic frequencies were estimated by gene counting [epsilon 2 = 0.01, epsilon 3 = 0.88, epsilon 4 = 0.11]. The Hardy-Weinberg expectation of genotype distribution was calculated from the estimated allele frequencies and a chi 2-test was used to test for equilibrium [chi 2 = 0.487, P>0.05]. Student's t-test indicated that subjects with the two most common apo E genotypes [E4/E4, E3/E3] were different from each other in means serum total cholesterol [t = 3.1, P<0.01]. Allele frequencies of the present study performed on Iranian subjects were similar to those of Japanese, Korean [Asian groups] Mexican and African American populations

Use of vectorette and subvectorette PCR for the isolation of terminal sequences from yeast artificial chromosome [YAC] clones

Development of yeast artificial chromosome [YAC] vectors, molecular cloning of large segments of chromosomal DNA, and their propagation in yeast cells has become feasible. Overlapping YAC provides a route to the development of physical maps of entire mammalian chromosomes. A rapid method was developed to isolate and sequence termini of YAC inserts quickly. The YAC clone is digested with a range of restriction enzymes, and ligated with a linker at its ends. The digested fragments were amplified using modified vector specific primers and a universal linker primer. PCR products were sequenced and the information used to drive new sets of primers for screening of YAC libraries to obtain overlapping clones and construct existing YAC contig

Lipoprotein and apolipoprotein metabolism among patients under chronic hemodialysis treatment

Pathogenic factors that may contribute to hypertriglyceridemia were studied in 55 patients who have been undergoing chronic hemodialysis treatment. Increased levels of triglyceride were observed in the patient group compared to normal controls [190 +/- 93 mg/dL vs. 121 +/- 45 mg/dL, p<0.05]. Similarly, augmented concentrations of VLDL-C were observed among patients compared to the control subjects [36 +/- 18 mg/dL vs. 24 +/- 8 mg/dL, p<0.05]. The major fraction of serum cholesterol was distributed in LDL and lesser amounts were measured in VLDL and HDL. The HDL-C fraction did not display significant variation in the patient group as compared to normal subjects. Apo B levels were higher in the patient group compared to normal subjects [97 +/- 26 mg/dL vs. 87 +/- 14 mg/dL, p<0.05]. In addition, apo A-I levels were significantly lower in the patient group compared to control subjects [84 +/- 13 mg/dL vs. 119 +/- 14 mg/dL, p<0.05]. A reverse relationship was observed between the concentration of plasma albumin and total cholesterol which may explain the reason for overproduction of lipids in renal failure, although other factors may contribute to the increased lipoprotein production observed in patients receiving chronic hemodialysis treatment