Constructs for protein expression in mammalian cell culture were inserted into the pCI-neo vector (Promega) with an N-terminal FLAG (DYKDDDDK) tag. Templates for in vitro transcription of radiolabeled mRNA were EcoRI/BamHI cloned into pGEM-4Z (Promega). MINX, MINX Δi and the expression vectors for FLAG-Y14, FLAG-eIF4A3, FLAG-CWC22 and their respective mutants were described previously (20 (link),24 (link)). CWC22 deletion mutants 110–908, 340–908, 1–769 and Δ669–759 were polymerase chain reaction (PCR) amplified and inserted into pCI-neo-FLAG. AdML-PT60 and AdML-PT60/e1(18) were generated by PCR amplification of a synthetic DNA. All constructs were verified by DNA sequencing.
Pgem4z
Manufactured by Promega
Sourced in United States
PGEM4Z is a plasmid vector commonly used in molecular biology laboratories for cloning and subcloning experiments. It contains an ampicillin resistance gene for selection, multiple cloning sites, and a lacZ gene for blue-white screening.
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Lab products found in correlation
Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (3 mentions)
Pci neo vector, by Promega (1 mentions)
Rabbit reticulocyte lysate, by Promega (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Mmessage sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
35s methionine cysteine, by PerkinElmer (1 mentions)
Bl21 de3 star plyss cells, by Thermo Fisher Scientific (1 mentions)
Ninta superflow affinity column, by Qiagen (1 mentions)
6 protocols using pgem4z
1
Mammalian Cell Protein Expression Protocols
2
In vitro import of mitochondrial precursor proteins
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Open reading frames (ORF) encoding mitochondrial precursor proteins were cloned into pGEM4Z (Promega). The desired ORFs were amplified by PCR using vector specific primers (M13_FORWARD and M13_REVERSE) and applied to in vitro transcription reactions using the mMessage SP6 transcription kit (Ambion). mRNA was isolated by LiCl precipitation according to the manufacturer’s instructions and applied to in vitro translation reactions using rabbit reticulocyte lysate (Promega) in the presence of [35S]-methionine/cysteine (Perkin-Elmer). Isolated mitochondria were incubated with translation products in import buffer (20 mM HEPES-KOH pH 7.4, 250 mM sucrose, 80 mM KOAc, 5 mM MgOAc, 10 mM sodium succinate, 1 mM DTT and 5 mM ATP) at 37°C for various times as indicated in the figure legends. Samples subjected to protease treatment were incubated on ice for 10 min with 50 μg/ml proteinase K (Sigma-Aldrich), followed by the addition of 1 mM PMSF and incubation for 10 min on ice. For analysis of protein complexes by blue native electrophoresis and antibody-shift mitochondria were solubilised in 1% digitonin (at 1 mg/mL) and incubated with the desired antibody for 30 min on ice, prior to clarification and subsequent analysis by BN-PAGE. Following in vitro import samples were separated by SDS-PAGE and BN-PAGE and radiolabelled proteins were detected by digital autoradiography.
3
Optimized Heterodimeric ZFN Constructs
4
Targeted Genome Editing with Obligate Heterodimeric ZFNs
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ZFNs targeting the CCR5 and AAVS1 loci have been described previously (11 (link),13 (link)). The following FokI variants were used to construct obligate heterodimeric versions of ZFNs (19 (link),20 (link)): EL:KK (CCR5) and ELD:KKR (AAVS1). An optimized pair of the AAVS1-targeting ZFNs were used in this study (Supplementary Figure S1). The ZFN coding sequences were cloned into a modified version of plasmid pGEM4Z (Promega, Madison, WI, USA) containing a sequence of 64 alanines 3′ of the inserted gene sequence (21 (link)), which was linearized by SpeI digestion to generate templates for mRNA synthesis. mRNA was prepared using the mMESSAGE mMACHINE® T7 ULTRA Kit (Life Technologies, Carlsbad, CA, USA) or by TriLink Biotechnologies (San Diego, CA, USA).
5
Optimized Heterodimeric ZFN Constructs
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ZFNs targeting the CCR5 and AAVS1 loci have been described previously28 ,61 (link). The following FokI variants were used to construct obligate heterodimeric versions of ZFNs62 : EL:KK (CCR5, experiments with mobilized blood CD34+ cells), ELD:KKR (CCR5, experiments with fetal liver CD34+ cells; AAVS1). An optimized pair of the AAVS1-targeting ZFNs was used in this study (Supplemental Fig. 14 ). The ZFN coding sequences were cloned into a modified version of plasmid pGEM4Z (Promega, Madison, WI) containing a sequence of 64 alanines 3′ of the inserted gene sequence63 (link), which was linearized by SpeI digestion to generate templates for mRNA synthesis. mRNA was prepared using the mMESSAGE mMACHINE® T7 ULTRA Kit (Life Technologies, Carlsbad, CA) or by TriLink Biotechnologies (San Diego, CA).
6
Recombinant Expression and Purification of B. miyamotoi GlpQ
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B. miyamotoi sensu lato glpQ from strain LB-2001 cloned into the prokaryotic expression vector pXT7 (18 (link)), a derivative of pGEM4Z and pSP64T (Promega, Madison, WI, USA), was transformed into BL21 Star (DE3)/pLysS cells (Invitrogen, Carlsbad, CA, USA), and transformants were used for protein production (6 (link)). The chromosome sequence for the protein is in GenBank (accession no. CP006647) (19 ). The 39.1-kDa recombinant GlpQ (rGlpQ) containing an N-terminal His tag was purified over an Ni-NTA Superflow affinity column (QIAGEN, Valencia, CA, USA) as described by the manufacturer. Purity was assessed by sodium dodecyl sulfate electrophoresis of �%^1 I1/4g of rGlpQ on a 4%�?"20% polyacrylamide gel and by Coomassie blue staining (Figure 1 ).
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