Production of correctly folded Fab antibody fragment in the cytoplasm of Escherichia coli trxB gor mutants via the coexpression of molecular chaperones.

Disulfide bonds are normally formed after a polypeptide has been exported from the reducing environment of the cytoplasm into a more oxidizing compartment, such as the bacterial periplasm. Recently, we showed that in Escherichia coli trxB gor mutants, in which the reduction of thioredoxin and glutathione is impaired, the redox potential of the cytoplasm becomes comparable to that of the mammalian endoplasmic reticulum, thus allowing the formation of disulfide bonds in certain complex proteins (P. H. Bessette et al., 1999, Proc. Natl. Acad. Sci. USA 96, 13703-13708]. Here, we investigate the expression of a Fab antibody fragment in the bacterial cytoplasm. The effect of coexpressing cytoplasmic chaperones (GroEL/ES, trigger factor, DnaK/J), as well as signal sequenceless versions of periplasmic chaperones (DsbC and Skp), was examined. Skp coexpression was shown to have the most significant effect (five- to sixfold increase) on the yield of correctly folded Fab. A maximum yield of 0.8 mg Fab/L/OD(600) Fab was obtained, indicating that cytoplasmic expression may be a viable alternative for the preparative production of antibody fragments.     

Thymus transplantation and disease prevention in the diabetes-prone Bio-Breeding rat

Bio-Breeding rat T lymphocytes proliferate poorly in response to alloantigen. Transplantation of Bio-Breeding rats with fetal thymus tissue from diabetes resistant rats leads to an improvement in the T cell proliferative response, but only if the thymus contains bone marrow-derived, radiation-resistant thymic antigen presenting cells of the diabetes-resistant phenotype. The current study provides evidence that thymus transplantation leading to the restoration of Bio-Breeding T cell proliferative function can also significantly reduce the incidence of insulitis and prevent the development of diabetes. It appears that a defect in the bone marrow-derived thymic APC population contributes to an abnormal maturation of Bio-Breeding T lymphocytes which in turn predisposes animals to insulitis and diabetic disease.     

Front Cover: Photoacoustic imaging of the human placental vasculature (J. Biophotonics 4/2020)

Successful therapy of twin‐to‐twin transfusion syndrome requires accurate imaging to guide laser photocoagulation of the anastomosing placental vessels. Photoacoustic (PA) imaging is an alternative imaging method that provides contrast for hemoglobin, and in this study, it was used to visualize chorionic superficial and subsurface vasculature in human placentas. The strong potential of PA imaging to guide minimally invasive fetal therapies was demonstrated. Further details can be found in the article by Efthymios Maneas, Rosalind Aughwane, Nam Huynh, et al. ( e201900167 ).

     

Characterization of purified leaf cytosolic pyruvate kinase from the C4 plant Cynodon dactylon

Cytosolic pyruvate kinase (EC 2.7.1.40) from leaves of the C₄ plant Cynodon dactylon (L.) Pers. was purified 56-fold to apparent homogeneity by polyethylene glycol fractionation and column chromatography including Q-Sepharose anion exchanger, ADP-Agarose and gel filtration. Nondenaturing PAGE of the final preparation resulted in a single protein band that co-migrated with the pyruvate kinase activity. Gel filtration and SDS-PAGE (± DTT) showed that this enzyme has a molecular mass of 200 kDa and is a homotetramer with a subunit molecular mass of 50 kDa. The subunits are not associated to each other with S-S bonds. The enzyme has a pH optimum of 6.2 and is heat stable. Typical Michaelis-Menten kinetics was obtained for both substrates, PEP and ADP, with K_m values of 64 and 235 μ M , respectively. Initial velocity studies indicated a sequential binding of the substrates to the enzyme.