The substitutions (Phe, Ala, Lys and Glu) were introduced into the cDNAs of the wt‐hPAH and the double truncated form ΔN102/ΔC24‐hPAH (catalytic domain) using the QuikChange® II site‐directed mutagenesis kit (Thermo Fisher Scientific Inc.). The pMAL‐hPAH 20 and pMAL‐ΔN102/ΔC24‐hPAH 21 plasmids, containing a cleavage site for factor Xa, were used as template, and the specific oligonucleotide primers listed in Table S1 were used for mutagenesis.
Quikchange 2 site directed mutagenesis kit
Manufactured by Thermo Fisher Scientific
The QuikChange II Site-Directed Mutagenesis Kit is a laboratory tool used to introduce specific mutations into double-stranded plasmid DNA. The kit provides a rapid and efficient method for site-directed mutagenesis without the need for subcloning, making it a versatile tool for various genetic engineering applications.
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3 protocols using quikchange 2 site directed mutagenesis kit
1
Site-Directed Mutagenesis of hPAH
2
NRBP1 cDNA Cloning and Mutagenesis
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The cDNA of Human NRBP1 was amplified from AW13516 cell line using Superscript III (Invitrogen, cat no 18080–093) in a TA cloning vector pTZ57R/T(InsTAclone PCR cloning kit, K1214, ThermoScientific), later site-directed mutagenesis was done using QuikChange II Site-Directed Mutagenesis Kit (cat.no. 200523) as per manufacturer’s instructions. Later both wild type and mutant NRBP1 cDNA sequenced confirmed using Sanger sequencing and were sub-cloned in to retroviral vector p BABE-puro using restriction digestion based cloning (SalI and BamHI).
3
Cloning and Expression of TtClpP and TtClpX
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TtClpP DNA was directly amplified from T. thermophilus HB8 DNA and cloned in to a pET41c (Novagen) expression vector using Ned I and Xho I restriction enzymes. The expressed gene contained the additional residues LEHHHHHHHH at its C terminus to allow affinity chromatography purification. Full-length ClpX DNA was first cloned into a pET28 vector using Ned I and Hind III restriction enzymes, resulting in residues MGSSHHHHHHSSGLVPRGSH added to the N terminus. While full-length ClpX was insoluble, similar to ClpX orthologs from other species, removal of its N-terminal domain (residues 1 to 54) allowed expression of a soluble ClpX construct (∆TtClpX) (48 (link)). Removal of the TtClpX N terminus was performed by GeneCust using Ned I and Hind III as restriction enzymes. TtClpP and ∆TtClpX were expressed in E. coli Bl21RIL cells following overnight expression at 20°C after induction with 1 mM isopropyl-β-d -thiogalactopyranoside (IPTG). A TtClpP mutant containing an S97A mutation was constructed using the QuikChange II Site-Directed Mutagenesis Kit from Thermo Fisher Scientific.
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