RDNA proteins

Fibrillar center Storage of inactive rDNA and proteins rDNA, RNA pol 1, UBF, topo I Scheer and Rose, 1984 Rendon et al., 1992 Raska et al., 1990b... 

Alternatively, some subunit viral vaccines can be generated by rDNA techniques and expressed in a continuous ceU line or insect ceUs. Recent advances in bioreactor design and operation have improved the successful production of IPV in large-scale bioreactors. However, roUer bottles or flasks are stiU used for most current vaccine production. Development of insect ceU culture will allow for very large-scale Hquid suspension culture (143). Several vaccine candidates such as gpl60 for HIV and gD protein for herpes have been demonstrated in the insect ceU culture system. However, no vaccine has been approved for human use. 

As reviewed by Ryffel (1996) most side effects in humans of a therapy with rDNA therapy may be predicted by data from experimental toxicology studies, but there are exceptions. IL-6 for example, induced a sustained increase in blood platelets and acute phase proteins, with no increase in body temperature. In human trials, however, there were increases in temperature. 

This approach appears somewhat irrational and without much scientific merit, since many of these new molecules are minimally toxic or nontoxic by this sort of acute evaluation. As in the case of interferons or monoclonal antibodies, the toxic effects observed in humans might not be predicted from safety assessments in rodents. An appropriate test species should be selected. Is the rat or mouse the appropriate species to evaluate a species-specific rDNA protein such as human growth hormone or interferons, or would nonhuman primates be more suitable Does the nonhuman primate really offer any advantages There is some consensus that the nonhuman primate may be a more appropriate species for testing some rDNA human proteins. 

The problem of the immunogenic nature of many human recombinant DNA proteins, and the potential to generate antibodies to a normal human protein, is of special interest to the immunotoxicologist. For example, 3 of 16 patients administered the rDNA-derived interferon-a (clone A) developed antibodies of the IgG class that were undetectable prior to or during therapy (Gutterman et al., 1982). These antibodies were capable of in vitro neutralization of interferon activity, although in vivo neutralization of interferon has not been documented. Since there are several different subtypes of interferon-a s, some contain epitopes not present on their own interferon subtype. Similarly, two patients treated with interferon-/ for many months developed high-titered antibody, which in one case was correlated with an inability of the patient s fibroblasts to produce interferon (Vallbracht et al., 1982). 

The exact mechanism of the immunogenicity of species-specific rDNA proteins is unknown, but is believed to be attributable to (1) the addition of extra amino acid residues during synthesis, which the host reads as foreign (2) denaturing of the native molecules or (3) contamination by E. coli polypeptides or lipopolysacchar-ides. 

A third consideration is that certain routes of administration may favor immu-nogenicity of recombinant proteins. In early trials, rDNA proteins introduced by subcutaneous or intramuscular injections (procedures known to improve the immu-nogenicity of proteins) resulted in a higher frequency of antibody responses than in the intravenous route. 

In summary, these are the clinically relevant questions about the immunogenicity of rDNA species-specific proteins will antibody be induced in the recipient that will neutralize the therapeutic effect or lead to immune complex disease What is the class (e.g., IgG or IgE) and specificity (i.e., reactivity against specific protein or contaminant) of the antibody induced The former antibody type could potentially neutralize the product and produce immune complex disease, while the latter could result in an anaphylaxis response. It is possible that the antibody induced is of insignificant health consequence, and its presence is known only because of improvements made in the sensitivity of detection methods with the introduction of the enzyme-linked immunosorbent (ELISA) assay. 

Description Aranesp is an erythropoiesis stimulating protein (EPO) produced using rDNA technology in CHO cells. It has 165 amino acids and the molecular weight is 30-37 kDa. 

This is the consequence of the traditional application of CE in the process and product monitoring of rDNA-derived biopharmaceuticals in biotechnological industries. However, related proteins, and dimer and related substances of higher molecular mass of somatropin, and aprotinin are evaluated by means of HPLC and size exclusion chromatography, respectively, by the EP. 

Protein polymers based on Lys-25 were prepared by recombinant DNA (rDNA) technology and bacterial protein expression. The main advantage of this approach is the ability to directly produce high molecular weight polypeptides of exact amino acid sequence with high fidelity as required for this investigation. In contrast to conventional polymer synthesis, protein biosynthesis proceeds with near-absolute control of macromolecular architecture, i.e., size, composition, sequence, topology, and stereochemistry. Biosynthetic polyfa-amino acids) can be considered as model uniform polymers and may possess unique structures and, hence, materials properties, as a consequence of their sequence specificity [11]. Protein biosynthesis affords an opportunity to completely specify the primary structure of the polypeptide repeat and analyze the effect of sequence and structural uniformity on the properties of the protein network. 

Recombinant DNA can be used to overcome a variety of potential problems in the industrial enzyme sector. A schematic outline of the types of problems encountered and the solutions by an rDNA program is shown in Figure 1. Basically, these problems can be divided into strict yield issues (making more of a given protein) and quality issues (making the product more useful). Because of the nature of the enzyme business, the highest return to the enzyme producer will usually come from increasing the value of the enzyme to the user. 

Figure 2. Efficiency of an rDNA approach to enzyme commercialization. Areas in boxes refer to the typical times required for Identification, Scale-up and Commercialization. Efficiencies are recognized in Identification of enzymes (protein engineering) and shortened Scale-up times (generic hosts).

Straight, A.F., Shou, W., Dowd, G.J., Turck, C.W., Deshaies, R.J., Johnson, A.D. and Moazed, D. (1999) Netl, a Sir2-assodated nucleolar protein required for rDNA silencing and nucleolar integrity. Cell, 97, 245-256. 

Products of rDNA technology are produced by genetic modification in which DNA coding for the required product is introduced, usually by means of a plasmid or a viral vector, into a suitable microorganism or cell line, in which that DNA is expressed and translated into protein. The desired product is then recovered by extraction and purification. 

IL-2, rDNA/Seragen [BIO] A-L-Methionyl-387-L-histidine-388-L-alanuie-l-388-toxin (Corynebacterium diphtheriae strain Cl) (388-2 ) protein with 2-133-interleukin 2 (human clone pTIL2-21a) [CAS] Interleukin-2 Fusion Protein [SY] Interleukin-2/diphtheria toxin fusion protein, recombinant [SY]... 

Lubiniecki AS, Lupker JH. Purified protein products of rDNA technology expressed in animal cell culture. Biologicals 1994 22 161-169. 

Other proteins which are being made using genetically engineered organisms include vaccines for animals and humans. Table 5.16 shows some examples of products that have used rDNA technology. 

---