Protein Engineering Techniques: Gateways to Synthetic by Krishna Mohan Poluri, Khushboo Gulati

By Krishna Mohan Poluri, Khushboo Gulati

This short offers a vast evaluation of protein-engineering examine, providing a glimpse of the commonest experimental equipment. It additionally provides quite a few computational courses with functions which are usual in directed evolution, computational and de novo protein layout. extra, it sheds mild at the merits and pitfalls of latest methodologies and destiny views of protein engineering techniques.

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8 In Vitro Homologous Recombination Imitating the natural in vivo recombination, in vitro homologous recombination methods require high sequence homology between the parental sequences. Several techniques were developed under in vitro recombination for synthesizing a variety of recombinant libraries (Fig. 6). The following sections will provide a glimpse of various important techniques formulated under this scheme. 1 DNA Shuffling DNA Shuffling is one of the first pioneering works in the era of recombination methods carried out by Stemmer and his colleagues for designing new proteins.

Further, Tang et al. has contributed an efficient and comparable method to the Trimer dimer method known as ‘small-intelligent’ library method (SILM). This method is capable to construct the small mutant libraries, devoid of inherent amino acid biases, stop codon, or rare codons of E. coli by combining the degenerate primers with appropriate PCR based mutagenesis method [61]. Other sophisticated methods in Focused mutagenesis include (a) synthetic saturation mutagenesis (can be combined with chip based DNA arrays) [62]; (b) Amber codon saturation mutagenesis (highly stable fluorinated proteins were obtained) [63, 64].

Used this method to heighten the ceftazidime resistance of TEM-1 β-lactamase [36]. 6 Targeting Glycosylases to Embedded Arrays for Mutagenesis (TaGTEAM) This method has been developed for targeted in vivo mutagenesis in yeast, which involved fusion of yeast 3-methyladenine DNA glycosylase (MAG1) to tetR DNA-binding domain and thereby increasing the mutation rates >800 fold in a specific region of DNA carrying tetO sites. Error prone homologous recombinations and error prone polymerase ζ were found to be the major contributors for point mutations in TAGTEAM [37].

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