Generation of anNco I restriction site for translational fusions using PCR-mediated, site-directed mutagenesis

1995 ◽  
Vol 13 (3) ◽  
pp. 232-242 ◽  
Author(s):  
Alfredo F. Galvez ◽  
Benito O. de Lumen
Keyword(s):  
Restriction Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Site-directed mutagenesis of multi-copy-number plasmids: Red/ET recombination and unique restriction site elimination

BioTechniques ◽  
2009 ◽  
Vol 46 (7) ◽  
pp. 527-533 ◽  
Author(s):  
Stephan Noll ◽  
Gabriele Hampp ◽  
Hanna Bausbacher ◽  
Natalia S. Pellegata ◽  
Harald Kranz
Keyword(s):  
Copy Number ◽  
Restriction Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Unique Restriction Site ◽  
Unique Restriction

scholarly journals Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site-directed mutagenesis for therapeutic protein production

2019 ◽  
Vol 24 (1) ◽  
pp. 8
Author(s):  
Achmad Rodiansyah ◽  
Riyona Desvy Pratiwi ◽  
Sabighoh Zanjabila ◽  
Asrul Muhamad Fuad
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Expression Vector ◽  
Restriction Site ◽  
Stop Codon ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth ◽  
Ecori Restriction

Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained.

scholarly journals An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

2009 ◽  
Vol 10 (6) ◽  
pp. 479-482 ◽  
Author(s):  
Bao-zhong Zhang ◽  
Xin Zhang ◽  
Xiao-ping An ◽  
Duo-liang Ran ◽  
Yu-sen Zhou ◽  
...  
Keyword(s):  
Restriction Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Screening

scholarly journals REMP software to introduce a screening REstriction site in site-directed Mutagenesis Primer

SoftwareX ◽  
2021 ◽  
Vol 16 ◽  
pp. 100881
Author(s):  
Madhavi Latha Yadav Bangaru ◽  
Ravi Kumar Medabalimi ◽  
Sobhan Babu ◽  
Nidhanapati K. Raghavendra
Keyword(s):  
Restriction Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

2013 ◽  
Vol 37 (3) ◽  
pp. 330
Author(s):  
Yanan WANG ◽  
Xudong LIU ◽  
Linlin MU ◽  
Zhipeng LIU ◽  
Chunmei LI ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Cynoglossus Semilaevis ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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