All eukaryotic expression vectors have the same basic format. The gene of interest, which may be equipped with sequences that facilitate the secretion and purification of the heterologous protein, is under the control of eukaryotic promoter, polyadenylation and transcription terminator sequences. To simplify both maintenance and recombinant DNA manipulations, eukaryotic expression vectors are routinely maintained in E. coli.
Several different fungal-based expression systems have been developed for the production of heterologous proteins. The yeast S. cerevisiae, which is well characterized genetically and can be grown in large fermenters, has been used extensively for this purpose. Both episomal and integrating expression vectors, as well as artificial chromosomes, have been constructed. However, with S. cerevisiae as the host cell, a number of recombinant proteins are hyperglycosylated, and in some cases, protein yields are low because the capacity of the cell to properly fold and secrete proteins has been exceeded. Other yeast and filamentous fungal systems have been developed for the production of heterologous proteins. Of these, the methylotrophic yeast P. pastoris has been used successfully because of the low occurrence of hyperglycosylation, the ease of obtaining high cell densities, and the rapid and strong response of the AOX1 promoter (usually used to drive the gene of interest) to methanol. A “humanized” strain of P. pastoris has been genetically altered to produce glycoproteins with glycosylation patterns that are identical to those found on the same proteins produced in human cells.
A large number of biologically active heterologous proteins have also been produced in insect cells grown in culture using baculoviruses to deliver the gene of interest into the insect host cell. This system is advantageous because posttranslational protein modification is similar in insects and mammals, and the baculoviruses used in these systems do not infect humans or other insect cells. The baculovirus most commonly used as a vector is AcMNPV. A gene of interest is inserted into the AcMNPV genome by homologous or site-specific recombination between sequences on a transfer vector carrying the target gene and the AcMNPV DNA. Recombination occurs either in insect cells doubly transfected with the transfer vector and viral DNA, in E. coli as an intermediate host, or in an in vitro reaction catalyzed by purified integration enzymes. The last two methods eliminate the need to identify and purify recombinant baculoviruses using plaque assays. Once the target gene has been inserted, recombinant AcMNPV DNA is introduced into insect cells for heterologous-protein production. Improved insect host cells have been developed through genetic engineering to increase protein yields and to ensure that target proteins are properly glycosylated. In addition to production of a single protein of interest, the baculovirus–insect expression system is particularly amenable to producing functional multimeric protein complexes, such as virus-like particles, which are effective vaccines.
Many therapeutic proteins that require a full complement of posttranslational modifications are now produced in cultured mammalian cells, such as CHO cells. Most of the vectors that have been developed to introduce foreign genes into mammalian cells are based on mammalian viruses, especially SV40. The viral genome has been altered to remove some viral genes required for replication and viral-protein production and to include suitable mammalian transcription and translation signals to drive expression of the cloned gene. Expression of chromosomally integrated target genes can be increased by altering the epigenetic state of the insertion site through histone acetylation or insertion of chromatin-relaxing DNA elements. A major challenge for production of high levels of heterologous proteins in mammalian cell lines is preventing cell death, which is often induced by the stressful conditions of large-scale bioreactors. Strategies to improve cell growth and protein yields include genetically engineering host cells to block the transcription factor that induces apoptosis, to prevent accumulation of toxic metabolites in the culture medium, and to increase expression of proteins required for proper protein folding and secretion.
Natural proteins are often not well suited for biotechnology applications. For example, an enzyme may unfold, and therefore be inactivated, at high temperatures employed in an industrial process, or a therapeutic protein may be short lived due to protease sensitivity necessitating administration of high, somewhat toxic, doses. Random or directed mutagenesis can be employed to alter the nucleotide sequence encoding a protein to improve its stability, activity, specificity, cofactor requirements, or protease resistance. Straightforward protocols have been developed to introduce nucleotide substitutions into a gene on an oligonucleotide primer by PCR. When the specific amino acids that contribute to a property are known in advance, the defined nucleotide changes can be introduced on an oligonucleotide by overlap extension or inverse PCR. When the amino acid changes that will result in the desired property of a protein are unknown, libraries of randomly mutated sequences can be generated by performing a PCR under conditions that increase the error rate or by employing degenerate oligonucleotide primers. Most of the mutations will decrease the function of the encoded protein and therefore the libraries must be screened to identify proteins with desired characteristics. Shuffling of DNA segments from two or more genes creates a large number of hybrid proteins that can also be screened for unique biological activity.
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