Recombination heterologous

Obtained from protein isolation, not heterologously expressed Determined by purified Cannabis extract Determined by recombinant proteins isolates... 

As discussed above, alternative recombinant DNA techniques are necessary to efficiently generate genome-scale clone sets. One alternative exploits the ability of the Vaccinia virus DNA topoisomerase I to both cleave and rejoin DNA strands with high sequence specificity (Shuman, 1992a Shuman, 1992b). In the reaction, the enzyme recognizes the sequence 5 -CCCTT and cleaves at the final T whereby a covalent adduct is formed between the 3 phosphate of the cleaved strand and a tyrosine residue in the enzyme (Fig. 4.1). The covalent complex can combine with a heterologous acceptor DNA that has a 5 hydroxyl tail complementary to the sequence on the covalent adduct to create a recombinant molecule (Shuman, 1994). 

Nitrilases convert nitriles to the corresponding carboxylic acids and NH3 through a cysteine residue in the active site [50]. Because of their high enantio- and regio-selectivity, nitrilases are attractive as green catalysts for the synthesis of a variety of carboxylic acids and derivatives (Figure 1.10) [51,52]. Recently, a number of recombinant nitrilases have been cloned and characterized heterologously for synthetic applications [50,53,54]. 

Murashima, K., Chen, C.-L., Kosugi, A. et al. (2002) Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes. Journal of Bacteriology, 184 (1), 76-81. 

Table 1. Recombinant bacteria harboring the heterologous PHA biosynthesis genes and synthesized PHA... 

A recombinant pseudomonad comprising a heterologous nucleic acid molecule which encodes one or more desulfurization enzymes. 

When recombinant proteins are produced in a heterologous system, there may potentially be differences between the final product and the natural molecule. Hence, for each new protein produced in alfalfa, a thorough analysis of the processing, folding, assembly and post-translational modification is conducted to ensure the conformity of the purified molecules. This section describes the analysis of alfalfa-derived... 

The expression of recombinant proteins in cells in which they do not naturally occur is termed heterologous protein production (Chapter 3). The first biopharmaceutical produced by genetic engineering to gain marketing approval (in 1982) was recombinant human insulin (tradename Humulin ), produced in E. coli. An example of a more recently approved biopharmaceutical that is produced in E. coli is that of Kepivance, a recombinant keratinocyte growth factor used to treat oral mucositis (Chapter 10). Many additional examples are provided in subsequent chapters. 

High levels of expression of heterologous proteins can be achieved in recombinant E. coli (Table 5.3). Modern, high-expression promoters can routinely ensure that levels of expression of the recombinant protein reach up to 30 per cent total cellular protein. 

Although capable of glycosylating heterologous human proteins, the glycosylation pattern usually varies from the pattern observed on the native glycoprotein (when isolated from its natural source, or when expressed in recombinant animal cell culture systems). 

In most instances, expression levels of heterologous proteins remain less than 5 per cent of total cellular protein. This is significantly lower than expression levels typically achieved in recombinant E. coli systems. 

Figure 6.2 A Likely purification procedure for tPA produced in recombinant E. coLi cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltra-tion step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed, as tPA is known to bind immobilized Lysine molecules. Adapted with permission from Bio/Technology (1993), 11, 351... 

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