High level expression of heterologous proteins in methylotrophic yeast Pichia pastoris.

Expression of human tumor necrosis factor-alpha (TNF) and four different TNF analogs has been studied in Pichia pastoris by utilizing the alcohol oxidase gene promoter. TNF expression level in certain transformants accounted for as much as 36% of the soluble protein. TNF expression was stably maintained during high cell density fermentation (100 g dry cell weight/liter) resulting in a TNF production level of 6-10 g/liter. TNF contained in P. pastoris cell lysates was biologically active as determined by its cytotoxic effect on murine L-929 fibroblast cells and the bioactivity was retained for at least 6 months in the lysates stored frozen at -20 degrees C in the presence of protease inhibitor PMSF. TNF expressed in P. pastoris was recognized by monoclonal antibodies prepared against recombinant Escherichia coli derived TNF. TNF purified from P. pastoris has the expected N-terminal amino acid sequence and specific activity of 10(7) units/mg protein. TNF analogs were also expressed at levels comparable to that of native TNF. Three of the four analogs were insoluble when produced in P. pastoris.

Generation and use of an antigen-specific hybrid to study B-cell function.

Several important events are known to occur following the crosslinking of membrane-associated immunoglobulin (mIg) on B-lymphocytes. Among these include the internalization and re-expression of mIg. We have addressed two questions regarding the re-expression of mIg following an antigen-induced clearance of this B-cell receptor. 1) How long does it take for a cell to re-express its receptors after the first or second exposure to antigen, and 2) is this re-expression dependent upon the synthesis of new mIg? These questions, which in the past have been addressed primarily with heterogeneous populations of cells and using anti-immunoglobulin instead of antigen, are vital to understanding B-cell activation as well as antigen processing events. In order to address these questions, we have produced and characterized an antigen-specific B-cell hybrid 2C3E1 that expresses a membrane-associated form of immunoglobulin. A hybrid specific for a charged hapten (phthalate) was selected to avoid the possibility of nonspecific hydrophobic interactions of the binding ligand with the plasma membrane. After establishing the presence of mIg biochemically and demonstrating the ability of phthalate-keyhole limpet hemocyanin to induce the clearance of the phthalate-specific receptor from the plasma membrane, it was determined that re-expression of antigen-specific receptors was detected within one hour and completed by six hours after the first or second pulse with antigen and that this re-expression did not require the synthesis of new receptors. This later finding is novel and suggests that B-cell receptors are re-utilized or that a presynthesized pool of mIg exists within the cytoplasm of these cells. During the characterization of the 2C3E1 cell line, it was determined that this hybrid also produced a secreted form of the phthalate-specific immunoglobulin (sIg). An unexpected subsequent observation was that sIg was found associated with the cell surface in addition to membrane Ig. A likely source of this unexpected surface-associated sIg was observed as a vesicle on the surface of many cells that is associated with a high concentration of sIg. This finding and its possible relevance to Ig secretion per se are considered further in the paper which follows this one.

Biochemical and ultrastructural characterization of a novel cell structure associated with immunoglobulin secretion in B-lymphocytes.

In the companion paper, it was established that a secretory form of immunoglobulin, sIg, is present at or near the cell surface. This unexpected occurrence of sIg was postulated to be due to the labelling of sIg which remains temporarily associated with the cell packaged in a vesicle which appears to bud from the plasma membrane at a single pole of the cell. The question that is addressed in this report is whether or not this polar accumulation of sIg represents a common pathway for the exit of this protein which is used by antibody-producing cells. This question is important since, in spite of the fact that the intracellular events associated with immunoglobulin synthesis (processing and movement between subcellular compartments) have been defined, very little data exists on how or where immunoglobulin finally leaves the plasma cell. This question was approached here by first demonstrating that the polar immunoglobulin secretory vesicles (ISV) are associated with several sIg-producing cells including other hybridomas, B-cell lines, and mitogen-activated spleen cells. The second approach was to characterize the ISV on the cell ultrastructurally and to establish that these vesicles are released from the cell carrying with them sIg. Isolated vesicles released from biosynthetically labeled Ig-producing cells were analyzed by SDS-PAGE in order to confirm the presence of sIg and to determine the number of other proteins associated with the ISV, their molecular weights, and the degree of disulfide crosslinking of the molecules comprising this structure. Finally, the kinetics of sIg release was established by a pulse chase protocol for biosynthetically labeled cells, and by monitoring the release of radioactive Ig from surface iodinated cells. As was predicted from our biochemical studies of the ISV, we observed a very slow phase of sIg release as well as a rapid release phase. Our studies have established that at least one of the pathways for the release of Ig from hybridomas, B-cell lines, and normal splenic B-cells is via a polar multivesiculated structure that we have termed ISV, and that the sIg can be released either as a free form of the protein or packaged within a satellite vesicle which may release the sIg later and perhaps at considerable distance from the cell that produced it.

Comparison of methyl-para-hydroxybenzimidate and 1,3,5-trichlorotriazine in producing sensitive target cells for use in the hemolytic spot assay.

Two bifunctional reagents were shown to be useful in the coupling of immunogens to the surface of either nucleated or non-nucleated cells. The heterobifunctional reagent methyl- para -hydroxybenzimidate was used to couple aromatic amino-haptens to the surface of SRBC which resulted in a stable and sensitive target cell capable of detecting as little as 20 pg of purified anti-hapten antibody in the hemolytic spot assay. The multifunctional reagent 1, 3, 5-trichlorotriazine yielded similar results when coupling amino-haptens to the surface of SRBC for use in the hemolytic spot assay. This reagent was also used to couple protein to the surface of SRBC which were able to detect as low as 1 ng of purified anti-protein antibody in the hemolytic spot assay. The sensitized SRBC produced using either of these coupling reagents were shown to remain stable for several months giving reproducible results from one test to another. Lastly, 1, 3, 5-trichlorotriazine was used to couple the hapten 4-aminophthalate to the surface of nucleated cells with retention of greater than 90% cell viability with continued growth and cellular division in culture.