[Engineering of a System for the Production of Mutant Human Alpha-Fetoprotein in the Methylotrophic Yeast Pichia pastoris].

A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

[Production of humanized F(ab')2 fragment of rabies blocking antibodies in Pichia pastoris yeast].

The yeast strain Pichia pastoris, a producer of humanized F(ab')2 fragments of rabies-blocking antibodies, has been obtained. Human chaperone BiP coexpression caused a twofold increase of the immunoglobulins secretion level. The use of Fos and Jun zippers in the composition of heavy chains facilitated the dimerization of F(ab')2 fragments of the shared pool of secreted immunoglobulins up to 75%.

[Enzyme activity of the formate hydrogenlyase complex in Citrobacter freundii]. / Aktivnost' fermentov formiatgidrogenliaznogo kompleksa u Citrobacter freundii.

Citrobacter freundii 62 can grow in the absence of oxygen in media containing glucose, peptone, fumarate or malate. When the medium contained fumarate or malate, the culture could grow under anaerobic conditions only in the presence of molecular hydrogen, formate or nitrate. The highest activity of formatehydrogenlyase and hydrogenase was found when C. freundii grew in a medium with glucose and formate. The activity was lower in media with other organic substrates, particularly, in the absence of formate or H2. The activity of hydrogenase was very low in cells grown under aerobic conditions or in the presence of nitrates while the activity of formatehydrogenlyase was not found at all for all practical purposes. The activity of formate dehydrogenase assessed in the presence of methylene blue was rather high irrespective of the conditions under which the culture was grown. However, when the activity of formate dehydrogenase was determined in the presence of benzyl viologen, it was high only in cells grown in the medium with glucose and formate.

[Cytochrome composition and content of Citrobacter freundii]. / Sostav i soderzhanie tsitokhromov u Citrobacter freundii.

Citrobacter freundii, strain 62, produces considerable amounts of cytochromes c and b when it grows in media with different carbon compounds under both aerobic anc anaerobic conditions. The content of cytochromes c increases under anaerobic conditions, particularly in the presence of formate or DMSO, and under aerobic conditions if formate is added. In contrast, the amount of cytochromes b decreased when the culture is grown in media with formate under anaerobic conditions. Cytochrome o is principal terminal oxidase in C. freundii. Under certain growth conditions, the microorganism, just as E. coli, produced also cytochromes a and d.

[Conditions for the formation of molecular hydrogen by Citrobacter freundii]. / Usloviia obrazovaniia molekuliarnogo vodoroda Citrobacter freundii.

Cell suspensions of Citrobacter freundii grown under anaerobic and aerobic conditions are capable of evolving molecular hydrogen. Hydrogen evolution is more active by the cells of anaerobic cultures in the exponential growth phase in the presence of reduced methylviologen or formate. The optimal temperature for hydrogen evolution from formate is about 40 degrees C, the optimal pH is 6.2 to 6.4. The presence of O2 in the gaseous phase inhibits hydrogen evolution by the cells; however the inhibition is incomplete and reversible. Nitrate also inhibits H2 formation from formate while fumarate does not inhibit the process.