pcDNA4/HisMax A, B, and C载体介绍： pcDNA4/HisMax is specifically designed to maximize protein expression in a variety of mammalian cells. The vector contains the QBI SP163 translational enhancer to increase expression levels two- to five-fold above those seen with the promoter alone . In addition to SP163-enhanced expression, pcDNA4/HisMax includes a cleavable N-terminal Xpress tag for rapid detection of recombinant protein with an Anti-Xpress Antibody. pcDNA4/HisMax is available TOPO Cloning-ready for five-minute cloning of Taqamplified PCR products.
pcDNA4/HisMax A, B, and C are 5.3 kb vectors derived from pcDNA4/His and designed for overproduction of recombinant proteins in mammalian cell lines. Features of the vectors allow purification and detection of expressed proteins. High-level stable and transient expression can be carried out in most mammalian cells. The vectors contain the following elements:
Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells
QBI SP163 translational enhancer for increased levels of recombinant protein expression (Stein et al., 1998) (see page 4 for more information)
Three reading frames to facilitate in-frame cloning with an N-terminal peptide encoding the Xpress epitope and a polyhistidine metal-binding tag
Zeocin resistance gene for selection of stable cell lines
Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g. COS-1, COS-7)
The control plasmid, pcDNA4/HisMax/lacZ, is included for use as a positive control for transfection, expression, and detection in the cell line of choice. 实验流程： Use the following outline to clone and express your gene of interest in pcDNA4/HisMax.
Consult the multiple cloning sites described on pages 5-7 to determine which vector (A, B, or C) should be used to clone your gene in frame with the N-terminal Xpress epitope and the polyhistidine tag.
Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50 to 100 μg/ml ampicillin or 25-50 μg/ml Zeocin.
Analyze your transformants for the presence of insert by restriction digestion.
Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in frame with the N-terminal peptide.
Transfect your construct into the cell line of choice using your own method of transfection. Generate a stable cell line, if desired.
Test for expression of your recombinant gene by western blot analysis or functional assay.
To purify your recombinant protein, you may use metal-chelating resin such as ProBond. ProBond resin is available separately. 表达目的基因： We have a wide variety of mammalian expression vectors utilizing the CMV or EF-1α promoter. Vectors are available with the Xpress (N-terminal), c-myc (C-terminal), or V5 (C-terminal) epitope for detection and either the neomycin, blasticidin, or Zeocin resistance genes. All vectors utilize the polyhistidine tag for purification using ProBond resin.
The pcDNA4/HisMax vectors are fusion vectors. To ensure proper expression of your recombinant protein, you must clone your gene in frame with the ATG at base pairs 1080-1082. This will create a fusion with the N-terminal polyhistidine tag, Xpress epitope, and the enterokinase cleavage site. The vector is supplied with the multiple cloning site in three reading frames relative to the N-terminal peptide to facilitate cloning.
If you wish to clone your gene as close as possible to the enterokinase cleavage site, follow the guidelines below:
Digest pcDNA4/HisMax A, B, or C with Kpn I.
Create blunt ends with T4 DNA polymerase and dNTPs.
Clone your blunt-ended insert in frame with the lysine codon (AAG) of the enterokinase recognition site.
