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. For antibody to the Xpress epitope, please see the next page.
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.
