Production of Recombinant Gene Products in Industry Help
Production of Recombinant Gene Products in Industry
Proteins with industrial, agricultural, or pharmaceutical applications can be produced in a variety of microbial or cell culture systems. Several host systems have been developed: bacterial (E. coli, Bacillus), fungal (yeasts, Aspergillus, Fusarium), plant, mammalian, and insect cell culture systems. In order to produce a desired gene product in one of these systems, the gene must first be cloned. If it is a eukaryotic gene, a complete cDNA clone (from start ATG to final stop codon) must be obtained, particularly if expression will take place in a bacterial system. Bacteria do not possess the machinery to splice introns, and other systems may or may not correctly carry out intron splicing of genes from significantly different organisms. Furthermore, bacteria cannot carry out posttranslational modifications, such as glycosylation, that may be required for protein function. The cDNA clone is spliced behind the control of a host-specific promoter in a plasmid vector to guide transcription in the host system. For example, a cloned human gene would be spliced behind the promoter from the yeast alcohol oxidase (AOX) gene for expression in a yeast host-vector system. The AOX gene promoter is a strong promoter (i.e., produces many mRNA molecules) and can be upregulated by the presence of methanol. Once this recombinant expression plasmid is created, it is introduced into the cells of the host system. Stable recombinant cells can be grown in the lab and then used to produce the desired gene product. Expression of a gene from an organism in a cell of the same species is called homologous gene expression. When a gene is expressed in a completely different host-cell system, the term heterologous gene expression is used. This is the method employed to produce a variety of useful recombinant proteins, such as human insulin, blood-clotting factors, and human growth hormone.
The Human Genome Project
Alarge collaborative effort to decode all of the 3 billion nucleotide base pairs of the human genome was completed in 2003, the 50th anniversary of the discovery of the structure of DNA by James Watson and Francis Crick. The international Human Genome Project effort was carried out and funded by government, coordinated by the National Human Genome Research Institute (NHGRI), and industry by Celera Genomics. It cost $3 billion for the public effort and $300 million for the private effort. Each of the government funded laboratories chose individual chromosomes to sequence while the industry effort sequenced the entire genome in a large "shotgun" approach using computers to assemble the data into a genome-wide map. All-in-all, over 20 billion bases of sequence information was gathered and analyzed. There is so much computer data that new hardware systems have been developed and storage space is measured in terms of terabytes (1015), which is one thousand times larger than a gigabyte (1012).
It is estimated that there are approximately 20,000 protein-coding genes within the 3 billion base pairs that make up the human genome. These genes are make up less than 2% of the total base pairs of DNA in the genome; the remaining DNA contains the information for RNA molecules, regulatory sequences, introns, repetitive DNA and other non-coding genetic elements (see Chapter 13). Human genes can range in size from thousands to hundreds of thousands of base pairs (including exons and introns). For example, analysis of sequence data from chromosome 22 reveals that it contains the genes within which mutations are responsible for at least 27 human disorders, including brain cancers and schizophrenia. It is estimated that chromosome 22 contains over 800 genes, the largest of which exceeds 500,000 base pairs. Of all of the protein-coding genes on this chromosome, only half (~400) have a hypothesized cellular function, as discovered through sequence database comparisons. Gene families, groups of genes that are similar, have been identified that appear to have originated from tandem duplication of genes and subsequent divergence by mutations. And that is one of the smallest of the 23 human chromosomes to be analyzed.
The completion of this human genome has ushered in a new era in genetic and medical research. It will take decades to interpret and understand all of the information represented by the A's, T's, G's, and C's in our DNA. Not only will scientists begin to better understand gene and chromosome structure and function, but patterns and interactions between genes willemerge. This information also brings with it ethical concerns over the use of genetic information by government agencies, medical institutions, and insurance companies. There is much to discuss and discover as we enter this new age.
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