Methods of Protein Engineering

determination of the 3D structure (if there are no similar structures available then heavy metal derivation is necessary)  

analysis of target amino acids to achieve improvement of desired characteristics (for changes in activity, knowledge of the mechanism is crucial)  

repetition of steps 7-10 if success was not achieved the first time. 

When protein engineering started to become a powerful tool to achieve new or enhanced characteristics, only limited methods were available. 

The key to changing an amino acid lies within the development of PCR and the exploitation of the ability to use primers which match parts of the target DNA. 

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It is possible to modify a natural enzyme, chemically or more commonly by the methods of protein engineering, in such a way that its specificity is altered, even to the point that the... 

The invention of new methods of protein engineering in the past decade has led to the construction of an abundance of P450 mutants with new tailored properties. Recent major achievements include significant increases in productivities, yields, and rates of catalytic turnover as well as modification of substrate specificity and efficient multistep reactions in whole-cell biocatalysts, coming one step closer to the technical application of cytochrome P450 monooxygenases. 

Some recent approaches to orientated immobilization exploited methods of genetic engineering to introduce suitable binding site like a cysteine residue [101]. Mansfeld and Ulbrich-Hofmann introduced cysteine on a thermolysinelike neutral protease from Bacillus stearothermophilus [114]. This approach is especially interesting since it allows the introduction of cystein residues in different positions of the protein molecule and therefore enables one to study the effects of molecule orientation on its biological activity [115]. More information about the oriented immobilization can be found in recent reviews of Turkova [101,110]. 

A better understanding of the photosynthetic units of the in vivo system are essential for both the in vitro and in vivo approaches but if one is looking for practical applications the emphasis of the work changes from one approach to the other. If one is to modify photosynthesis in vivo one is almost certainly going to use the methods of genetic engineering so that immediately one needs to know the specific protein complexes involved, their sequences and those of their genomes. 

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