Recombinant Methods

Recombination in a genetic sense means the breaking and rejoining of DNA in new combinations. Such a process is crucial for all living species, because it significantly speeds up the Darwinian process ( sexual evolution ). When mimicking this process in the laboratory, the presence of a set of recombination enzymes, which ensure the 

Interesting new homology-dependent and -independent methods have been developed that are not discussed in detail here, including RDA-PCR (55), MURA 54), RETT (55), ITCHY (56), SISDC (57), SCOPE 58), SCATCHY 59), and SHIPREC 60). These and other methods have been reviewed critically by Lutz and Patrick (57). A novel method based on random insertion and deletion of arbitrary number of bases for codon-based random mutation (RID) was developed by Sisido, which introduces additional amino acids or deletes them in the WT 61). Of special note is the fact that some non-natural amino acids can also be introduced, although this process is more complicated. 

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Vaccine candidates are based on the two viral surface proteins, gD and gB (80). Recombinant methods are used to express the proteins, either in Chinese hamster ovary (CHO) cells or in baculovims. The proteins are purified as subunits and formulated with different adjuvants. Clinical trials with these vaccine candidates have been performed, but the results to date have not been encouraging. 

Combinatorial Chemistry. Figure 2 Chemical libraries are prepared either by parallel synthesis or by the split-and-recombine method. In the latter case, coupling m building blocks in m separated reaction flasks through n synthetic cycles on a beaded polymer carrier generates a combinatorial library with nf individual compounds and one compound per bead. 

Ciawi E, Ashokkumar M, Grieser F (2006) Limitations of the methyl radical recombination method for acoustic cavitation bubble temperature measurements in aqueous solutions. J Phys Chem B 110 9779-9781... 

Derewenda ZS. 2004. The use of recombinant methods and molecular engineering in protein crystallization. Methods 34 354-363. 

More successfully, the (S)-Hnl from Manihot esculenta has also been overexpressed in E. coli [41] and the lysate of the transformed cells showed an enzyme activity of 0.5 units per ml of the culture. A culture of 801 volume of the recombinant MeHnl followed by a short purification procedure [41] yielded 40,000 U. To obtain the equivalent amount of enzyme from the parent plant material would require the processing of 100 -200 kg of dried cassava leaves and thus this recombinant method for the production of MeHnl is a significant practical development. Hence, this recombinant MeHnl has allowed a study of (S)-cyano-hydrin production to be performed [41]. 

This operation is illustrated by work on the human cytokine GCSF, a protein of 174 residues. The disulfide-linked loop (64-74) of this protein, thought to be involved in binding to the receptor, was replaced by various non-coded synthetic structures.[10] In this case recombinant methods were used to produce a variant of the parent protein having the appropriate cleavage sites for the production of the wanted fragments. 

Biotechnology for medical applications had become widely accepted and recombinant methods were familiar to most scientists working in the field. The first German biotech companies, the pioneers of the biotech industry in Germany, Qiagen (established 1984), Rhein Biotech (established 1986), MWG-Biotech (established 1990)and Evotec (established 1993), were struggling along. 

For the purpose of synthesizing flavor peptides or proteins in large scale, we developed "protein recombination method" and "enzymatic synthesis using chemically modified enzyme". "Protein recombination method" was applied to the synthesis of C-terminal portion of p-casein and its analog. Chymotrypsin was chemically modified by Z-DSP in aqueous solution. It was stable for organic solvents. Using this modified enzyme, we succeeded in the synfiiesis of Inverted-Aspartame-Type Sweetener "Ac-Phe-Lys-OH" in one step. 

Today, it is well-known that peptides or proteins exhibit various kinds of taste. Our group has been researching on the relationship between taste and structure of peptides, BPIa (Bitter peptide la, Arg-Gly-Pro-Pro-Phe-Ile-Val) (7 as a bitter peptide, Om-p-Ala-HCl (OBA), Om-Tau-HCl as salty peptides(2j, and "Inverted-Aspartame-Type Sweetener" (Ac-Phe-Lys-OH) as a sweet peptide(5). The relationship between taste and chemical structure was partly made clear. Since commercial demand for these flavor peptides is increasing, we need to develop new synthetic methods which can prepare these peptides in large scale. We developed the following two methods (1) protein recombination method as a chemical method, (2) enzymatic synthesis using chemically modified enzyme as a biochemical method. 

After screening about 10000 individual variant clones, more than 60 primary hit clones with more than 50% increased stability were selected and recombined using a proprietary recombination method [28]. After two rounds of recombination and screening with increased CIAA concentrations, ten clones were selected for retesting on shake-flask scale. 

The volume of the gas in the reservoir may be calculated using the compressibility equation of state. This calculation is based on 1 lb mole of gas using Equation 3-39. The composition of the gas in the reservoir calculated by the recombination method can be used to compute the pseudocritical properties so that the compressibility factor may be obtained in the same manner as illustrated in Examples 3-10 and 3-12. 

The recombination method gives results as accurate as laboratory results if accurate values of compositions and gas-oil ratios are available. 

The efficiency of S tEP recombination is similar to that of other in vitro recombination methods, but an advantage of this method lies in the fact that the reaction can be carried out in a single test tube. Template DNA can be double-stranded as well as single-stranded, and there is no significant restriction to the number of parent templates to be recombined. 

Table 11.2 Selected DNA mutagenesis and recombination methods (Kurtzman, 2001).

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