Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells.

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed.

Extremely thermostable serine-type protease from Aquifex pyrophilus. Molecular cloning, expression, and characterization.

A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 degreesC, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.

Random sequence analysis of genomic DNA of a hyperthermophile: Aquifex pyrophilus.

Aquifex pyrophilus is one of the hyperthermophilic bacteria that can grow at temperatures up to 95 degrees C. To obtain information about its genomic structure, random sequencing was performed on plasmid libraries containing 0.5-2 kb genomic DNA fragments of A. pyrophilus. Comparison of the obtained sequence tags with known proteins revealed that 123 tags showed strong similarity to previously identified proteins in the PIR or Genebank databases. These included three proteases, two amino acid racemases, and three enzymes utilizing oxygen as substrate. Although the GC ratio of the genome is about 40%, the codon usage of A. pyrophilus showed biased occurrence of G and C at the third position of codons, especially those for amino acids such as asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, and tyrosine. A higher ratio of positively charged amino acids in A. pyrophilus proteins as compared with proteins from mesophiles suggested that Aquifex proteins might contain increased ion-pair interaction that could help to maintain heat stability.

The production of L-serine with a methylotrophic microorganism using the L-serine pathway and coupling with an L-tryptophan-producing process.

The cultural conditions for the production of enhanced formation of L-serine (up to 7 g/L) are described with the methylotrophic bacterium Pseudomonas 3 ab (DSM 672). The batch process is divided into three parts: (1) the biomass production phase, (2) substrate limitation period, and (3) L-serine accumulation phase. The initial specific production rate of q(p) = 0.1 g L-serine/g dry wt/h is based on the inhibition of the L-serine pathway. This is accomplished by high precursor concentrations (glycine) and a pH shift to pH 8.5. The enzymatic background is discussed. Furthermore, a coupling of the L-serine process with a L-tryptophan-producing process is demonstrated.

Continuous production of L-tryptophan from indole and L-serine by immobilized Escherichia coli cells.

Escherichia coli B 10, which has high activity of tryptophan synthetase, was grown in a 50-L batch culture in order to determine in which growth phase the cells have the highest specific tryptophan productivity. Accordingly, whole cells of the stationary phase were used for immobilization in polyacrylamide beads. After immobilization, these immobilized cells had 56% activity of tryptophan synthetase compared with that of free cells. First, the properties of immobilized cells were investigated. Next, discontinuous productions of L-tryptophan were carried out by using immobilized cells. In discontinuous production of L-tryptophan by the batch, the activity remaining of immobilized cells was 76-79% after 30 times batchwise use. In continuous production of L-tryptophan with a continuous stirred tank reactor (CSTR), the activity remaining of the immobilized cells was 80% after continuous use for 50 days. The maximum productivity of L-tryptophan in this CSTR system was 0.12 g tryptophan L(-1) h(-1).

Production L-tryptophan by Escherichia coli cells.

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.