Other Protein Hosts

The overproduction of a wide variety of proteins has now been achieved in . coli and other cloning hosts. Many of these proteins are in chnical trials and, as indicated earlier, over a dozen are already on the market. The current status of many of these proteins is summarized in Table 24.2. The efficacy of mai r of the proteins listed remains to be determined because until the advent of recombinant DNA technology sufficient quantities were not available to enable clinical trials to be undertaken. It should be noted that clinical efficacy alone is not sufficient. Maiket size isjust as important since it can cost up to 50 million to bring a new dmg to the market place and company shareholders expect a good return on their investment. 

Virus infection obviously upsets the regulatory mechanisms of the host, since there is a marked overproduction of nucleic acid and protein in the infected cell. In some cases, virus infection causes a complete shutdown of host macromolecular synthesis while in other cases host synthesis proceeds concurrently with virus synthesis. In either case, the regulation of virus synthesis is under the control of the virus rather than the host. There are several elements of this control which are similar to the host regulatory mechanisms, but there are also some uniquely viral regulatory mechanisms. We discuss various regulatory mechanisms when we consider the individual viruses later in this chapter. 

Other proteins, such as those needing posttranslational modification for their correct folding or activity, still depend on alternative expression systems. In addition, hosts which are suitable... 

Abstract This chapter updates but mostly supplements the author s Ange-wandte Review,111 setting in context recent advances based on protein and nucleic acid engineering. Systems qualify as a true enzyme mimics if there is experimental evidence for both the initial binding interaction and catalysis with turnover, generally in the shape of saturation kinetics. They are discussed under five broad headings mimics based on natural enzymes, on other proteins, on other biopolymers, on synthetic macromolecules and on small-molecule host-guest interactions. 

Mimics based on natural enzymes Mimics based on other proteins Mimics based on other biopolymers Mimics based on synthetic macromolecules Mimics based on small-molecule host-guest interactions... 

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

Recombination Requires a Host of Enzymes and Other Proteins... 

A second Drosophila transposon called mariner630 typifies the mariner / Tel transposon superfamily, which also contains members from nematodes,631 other invertebrates, fishes,632 amphibia,633 and possibly human beings.634 These transposons encode a transposase containing a D, D, D or D, D, E motif630 but no other proteins. They contain short 30-bp terminal inverted repeats and become inserted into host TA sequences.631 Movement of some repetitive sequences of the LINE635 and SINE636 families within the human genome may be assisted by mariner transposons.637... 

Recombinantproteins thatare notexpressed in inclusion bodies either will be soluble inside the cell or, if using an excretion vector, will be extracellular (or, if E. coli is the host, possibly periplasmic). They can be purified by conventional means. In some systems, expression is so good that the desired product is the major protein present and its purification is relatively simple. In systems where the expression level is low, the purification process can be tedious, though easier, it is hoped, than isolation from the natural source. It should be remembered that a procedure developed for isolating a protein from natural sources may not work successfully with the recombinant product, because the nature of the other proteins present influences many fractionation procedures. 

The interferons are a family of proteins secreted by animal cells in response to viral and parasitic infections, and are part of the host s defence mechanism. They display multiple activities, affecting the functioning of the immune system, cell proliferation, and cell differentiation, primarily by inducing the synthesis of other proteins. Accordingly, they have potential as antiviral, antiprotozoal, immunomodulatory, and cell growth regulatory agents. 

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