R-prime plasmids from Bradyrhizobium japonicum and Rhizobium fredii

The formation of R-prime plasmids was selected in crosses involving soybean microsymbionts with genomic Tn5 insertions and carrying plasmid pJB3JI (with one IS2) copy as donors and Escherichia coli HB101 as recipient. Whereas the parent plasmid was 60 kb, recombinant plasmids between 76 kb and 121 kb were obtained. Restriction and Southern analyses confirmed the mobilization of Tn5 on four R-primes from Bradyrhizobium japonicum I-110 and on an R-prime plasmid from Rhizobium fredii HH303. The largest R-prime plasmid was obtained from the rescue of two symbiotically defective R. fredii mutant strains that required adenosine.Non-standard abbreviation TDP transposon donor pool Scientific article number A-4728 and contribution number 7724 of the Maryland Agricultural Experiment Station     

In vivo suppression of phytochrome aggregation by the GroE chaperonins in Escherichia coli

When expressed in Escherichia coli, a truncated form of phytochrome (oat PHYA AP3 residues 464-1129) self associates to form a series of products ranging in size from monomers to aggregates of greater than 20 subunits. When these same phytochrome sequences are coexpressed with the chaperonins GroEL and GroES, the truncated phytochrome migrates as a native-like dimer in size exclusion chromatography and no higher-order aggregates were detected. GroEL and GroES inhibition of phytochrome aggregation in E. coli presumably occurs via the suppression of folding pathways leading to incorrectly folded phytochrome.     

Expression of Aspergillus niger N5-5 in E. coli and purification and identification of products

Due to the feature of high hydrolysis, tannase is widely used in food, beverage, brewing and other fields. However, high cost in producing natural tannase makes it difficult to apply tannase to industry in a large-scale. Microbial expression systems can be used for preparing numerous amount of enzyme at low cost, so in this paper Aspergillus niger N5-5 was expressed using E. coli system. Specific primers were designed based on the Aspergillus niger N5-5 sequence N3 (GenBank, No.: KP677552), and tannase gene tan was promoted to carry 6 His tag and enzyme cutting site which contains NdeI/HindIII using PCR amplification. Then, tannase gene tan was connected to expression vector by NdeI/HindIII enzyme cutting. In this way, recombinant expression vector tan-pET43.1a was formed. Then, the expression vector pET43.1a by NdeI/HindIII enzyme cutting was transformed into E. coli BL21 (DE3) to induce expression of Aspergillus niger N5-5. When the induced fungi were disrupted by the ultrasonic wave, the crude enzyme was extracted and purified by using the IMAC, and then the activity of the crude enzyme and pure enzyme was determined. According to the results of determination of the tannase activity, the tannase activity of the crude enzyme was greatly improved after the crude enzyme was purified, and the specific activity of the pure enzyme was about 8 times of that of the crude enzyme. The results of SDS-PAGE of the pure enzyme showed that the molecular mass of the pure enzyme was about 65 kDa/64–65 kDa, which was consistent with the expected result (64.2 kDa), It can be concluded that the crude enzyme solution was purified successfully. The results of pure enzyme’s protein identification by Western Blotting showed that clear protein bands pro-3 were observed. Molecular mass of clear protein bands pro-3 was about 65 kDa, which was in line with the expected results (64.2 kDa). It can be seen that the aforementioned expression protein could be specifically combined with His tag. It proved expression protein to be a recombinant fusion protein with 6 His tag.     

Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.