NDLI: SfiNX: a method for assembly of protein coding sequences with high success rates

Content Provider	Springer Nature Link
Author	Takai, Kazuyuki Hisamatsu, Keigo
Copyright Year	2016
Abstract	Concatenation of two NdeI–XhoI gene fragments via an oligonucleotide linker on a plasmid vector with an SfiI site was performed to evaluate success rates in construction of polycistronic genes expressible in Escherichia coli.A series of plasmids with an SfiI site between the selection marker and the replication origin were constructed. The three wheat eEF1B subunit genes inserted between the NdeI and XhoI sites of pET-22b were transferred to the SfiI-containing plasmid with a spectinomycin-resistance gene. Then, the marker gene in the resultant plasmids was substituted with the ampicillin-resistance gene. These plasmids were used for concatenation of two different genes via a linker oligonucleotide containing a ribosome-binding site. During these operations, 42 clones were picked up out of which 41 had the intended product plasmid.This method, named as the SfiNX method, is useful for trial-and-error based testing of different combinations of fusion and co-expression partners for optimization of recombinant protein production.
Starting Page	773
Ending Page	778
Page Count	6
File Format	PDF
ISSN	01415492
Journal	Biotechnology Letters
Volume Number	38
Issue Number	5
e-ISSN	15736776
Language	English
Publisher	Springer Netherlands
Publisher Date	2016-01-12
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Escherichia coli Fusion Gene assembly Polycistron Rare cutter Microbiology Biotechnology Applied Microbiology Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Bioengineering Applied Microbiology and Biotechnology Biotechnology

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