Class I MHC Tetramer Preparation: Expression plasmids
The basic strategy for production of class I MHC/peptide complexes from denatured subunits produced in E. coli was originally defined by Garboczi and Wiley in 1992. For tetramer production, the heavy chain is fused to a BirA substrate peptide (BSP•41), derived from the libraries constructed and screened by Schatz for sequences that are biotinylated by the E. coli enzyme BirA. Both the heavy chain and b2m subunits are expressed as insoluble inclusion bodies, which are solubilized with common denaturants such as urea or guanidium chloride prior to folding in vitro. Many different E. coli promoters will drive expression to high enough levels to result in inclusion bodies. For expression of the b2m subunit, we still use the original plasmid in the pHN1 vector described by Garboczi and Wiley; this plasmid uses a hybrid trp/lac promoter (the so-called tac promoter), which is IPTG or lactose inducible. In contrast, for expression of the class I heavy chains, we have replaced Garboczi and Wiley's pHN1-based vector with any of a variety of pET vectors (Novagen), which use the powerful T7 expression system pioneered by Studier and colleagues, simply because the pET vectors are more widely available and somewhat more convenient for subcloning new MHC genes.
Detailed protocols for the basic molecular biology techniques required to subclone new MHC alleles into our vectors are beyond the scope of this web page; these techiques are commonplace in nearly all modern immunology laboratories. The purpose of this page is to provide a brief description of our vectors, and the simple strategies that we employ for subcloning new MHC alleles.
Expression plasmids
The plasmids that we have built for expression of class I MHC heavy chains are nearly all based upon the plasmid pET24a+ from Novagen.
The basic features of pET24a(+) are as follows:
- The origin of replication is based upon pBR322, and results in copy numbers that are between pACYC-based plasmids and pUC or pBluescript-based plasmids.
- Kanamycin is used for selection of transformed E. coli. Most manuals recommend concentrations of kanamycin in the range of 25-40 µg/ml. In contrast, we have adopted recommendations of F. W. Studier, and use kanamycin concentrations of 100 µg/ml.
- An f1 origin is provided, permitting production of single-stranded DNA in a bacteriophage. This feature is in some sense a remnant of the days when both mutagenesis and sequencing required production of single-stranded DNA, so-called phagemid technology. Single-stranded DNA is no longer required for these purposes.
- The promoter that drives expression of the recombinant heavy chain is a hybrid T7-lac promoter. In the presence of the lac-Iq repressor, the T7-lac hybrid provides lower basal levels of expression compared to the original T7 promoter.
- The lac-Iq repressor is expressed from a constitutive promoter. This provides the protein that binds to the lac operator in the T7-lac hybrid promoter, and that results in better suppression of protein expression in the absence of the IPTG or lactose inducer.
- At the 5' end of the expression cassette, we use either the NdeI or NheI restriction sites for cloning. If the MHC allele of interest lacks an Nde I site, we use it for cloning; otherwise, we use the NheI site.
- At the 3' end of the expression cassette, we have inserted the sequence for the BirA substrate peptide (BSP), described below. We have prepared two versions of our basic vectors, with and without the His•tag fusion following the BSP.
pTCF33: a vector for expression of proteins fused to BSP41
The plasmid pTCF33 is designed for cloning genes to produce protein fusions to BSP41. It is based upon pET24a+, and has the following additional features:
- In order to produce C-terminal fusions, the 3' end of the construct contains
the coding sequence for BSP41, in frame behind Kpn I and BamH
I restriction sites. The BSP41 sequence is followed by a stop codon. These
sequences were cloned in at the Hind III site.
If your gene of interest does not contain a BamH I site, then you should clone into the BamH I site. Otherwise, you should clone into the Kpn I site.
- An irrelevant 2kb stuffer fragment is present upstream of the Kpn I site. This is solely designed to facilitate screening of subsequent recombinant plasmids.
- At the 5' end, the 2kb stuffer fragment is cloned in behind Nde I
and Nhe I sites.
If your gene of interest does not contain a Nde I site, then it is preferred for cloning, as it does not result in additional amino acids at the N-terminus of the protein (other than the initiator methionine). However, cloning into Nde I sites is somewhat more difficult than cloning into many other restriction sites, and some investigators may prefer the Nhe I site. Of course, if your gene contains an Nde I site, then you should clone into the Nhe I site.
We have prepared two variants of pTCF33—pTCF33.HisV1 and pTCF33.HisV2—that contain His6 tags follwoing the BSP. We haven't used these vectors much, and the His tag is generally not required for purification. We are investigating His-tagged variants for rare cases where proteolysis of the BSP is a significant problem that is not solved by standard methods. Please note that we have found that metal ion affinity columns are not compatible with the arginine folding buffer, so that theis technique cannot be used for affinity purification directly from the folding reaction.
Class I MHC Expression Plasmid Table
| PLASMID | MHC I Allele | BSP | VECTOR | ANTIBIOTIC | Circular Map | Seq | Vector NTI |
|---|---|---|---|---|---|---|---|
| pTCF33 | None | 41 | pET24a+ | Kan | txt | gb | |
| pTCF33.HisV1 | None | 41 | pET24a+ | Kan | txt | gb | |
| pTCF33.HisV2 | None | 41 | pET24a+ | Kan | txt | gb | |
| pTCF3 | H-2D(d) | 41 | pET24a+ | Kan | txt | gb | |
| pTCF30 | H-2D(k) | 41 | pET24a+ | Kan | txt | gb | |
| pTCF23 | H-2D(q) | 41 | pET24a+ | Kan | txt | gb | |
| pTCF20 | H-2K(d) | 41 | pET24a+ | Kan | txt | gb | |
| pTCF5 | HLA A*0101 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF28 | HLA A*0201-His | 41 | pET24a+ | Kan | txt | gb | |
| pTCF9 | HLA A*0205 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF48 | HLA A*1101 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF46 | HLA A*1101mut | 41 | pET24a+ | Kan | txt | gb | |
| pTCF11 | HLA A*2301 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF6 | HLA A*2402 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF7 | HLA B*0702 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF10 | HLA B*0801 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF37 | HLA B*3501 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF29 | HLA B*5701-His | 41 | pET24a+ | Kan | txt | gb | |
| pTCF35 | HLA Cw*0304 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF57 | HLA Cw*0602 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF31 | HLA E*01033 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF14 | Mamu B*17 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF24 | Patr A*0101 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF49 | Patr A*0301 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF8 | Patr A*0401 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF50 | Patr A*0501 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF44 | Patr A*0701 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF51 | Patr A*0901 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF52 | Patr A*1401 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF15 | Patr B*0101 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF22 | Patr B*0301 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF53 | Patr B*0302 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF39 | Patr B*0401 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF40 | Patr B*0501 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF54 | Patr B*0802 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF16 | Patr B*1301 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF45 | Patr B*1601 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF55 | Patr B*1602 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF56 | Patr B*1701 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF18 | Patr B*2301 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF19 | Patr B*2401 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF1 | Mouse HFE | 41 | pET24a+ | Kan | txt | gb | |
| pTCF12 | Human HFE | 41 | pET24a+ | Kan | txt | gb | |
| pTCF34 | MIC A*004 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF32 | MIC A*008 | 41 | pET24a+ | Kan | txt | gb | |
| pTCF26 | RT BM1 | 41 | pET24a+ | Kan | txt | gb |