An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes

2015 ◽  
Vol 476 ◽  
pp. 26-28 ◽  
Author(s):  
Zhiqiang Zhang ◽  
Kun Xu ◽  
Ying Xin ◽  
Zhiying Zhang
Keyword(s):  
Efficient Method ◽  
Restriction Enzymes ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site

scholarly journals The consequences of deglycosylation of recombinant intra-melanosomal domain of human tyrosinase

2017 ◽  
Vol 399 (1) ◽  
pp. 73-77 ◽  
Author(s):  
Monika B. Dolinska ◽  
Yuri V. Sergeev
Keyword(s):  
Oculocutaneous Albinism ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Insect Larvae ◽  
Melanin Biosynthesis ◽  
Human Tyrosinase

AbstractTyrosinase, a melanosomal glycoenzyme, catalyzes initial steps of the melanin biosynthesis. While glycosylation was previously studiedin vivo, we present three recombinant mutant variants of human tyrosinase, which were obtained using multiple site-directed mutagenesis, expressed in insect larvae, purified and characterized biochemically. The mutagenesis demonstrated the reduced protein expression and enzymatic activity due to possible loss of protein stability and protein degradation. However, the complete deglycosylation of asparagine residuesin vitro, including the residue in position 371, interrupts tyrosinase function, which is consistent with a melanin loss in oculocutaneous albinism type 1 (OCA1) patients.

scholarly journals Efficient method for site-directed mutagenesis in large plasmids without subcloning

PLoS ONE ◽  
2017 ◽  
Vol 12 (6) ◽  
pp. e0177788 ◽  
Author(s):  
Louay K. Hallak ◽  
Kelly Berger ◽  
Rita Kaspar ◽  
Anna R. Kwilas ◽  
Federica Montanaro ◽  
...  
Keyword(s):  
Efficient Method ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Large Plasmids

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue

Biochemistry ◽  
1989 ◽  
Vol 28 (2) ◽  
pp. 510-516 ◽  
Author(s):  
A. Martinez del Pozo ◽  
M. Merola ◽  
H. Ueno ◽  
J. M. Manning ◽  
K. Tanizawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Spectroscopic Properties ◽  
Site Directed Mutagenesis ◽  
Tryptophan Residue ◽  
Directed Mutagenesis ◽  
Multiple Site

scholarly journals Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

2017 ◽  
Author(s):  
Christopher Cozens ◽  
Vitor B. Pinheiro
Keyword(s):  
Total Population ◽  
Cost Effective ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Multiple Site ◽  
Biotechnological Applications ◽  
Wild Type ◽  
High Quality ◽  
Working Day ◽  
Library Transformation

ABSTRACTEngineering proteins for designer functions and biotechnological applications almost invariably requires (or at least benefits from) multiple mutations to non-contiguous residues. Several methods for multiple site-directed mutagenesis exist, but there remains a need for fast and simple methods to efficiently introduce such mutations – particularly for generating large, high quality libraries for directed evolution. Here, we present Darwin Assembly, which can deliver high quality libraries of over 108 transformants, targeting multiple (> 10) distal sites with minimal wild-type contamination (lower than 0.25% of total population) and which takes a single working day from purified plasmid to library transformation. Darwin Assembly uses commercially available enzymes, can be readily automated, and offers a cost-effective route to highly complex and customizable library generation.

scholarly journals A simple and efficient method for in vitro site-directed mutagenesis

2020 ◽  
Author(s):  
Dave Palis ◽  
Frank Huang
Keyword(s):  
Efficient Method ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Citation Statistics

This paper has been withdrawn by bioRxiv because its content, including the author names, was fabricated and fraudulently submitted in what may have been an attempt to game citation statistics or other metrics.

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