Focus: Expression Vector Subcloning

Transfer the HaloTag® Coding Region to Your Expression Vector of Choice

The HaloTag® 7 coding sequence is available in the Flexi® Vectors for expression in different in vitro and in vivo systems. There may be times when expression is needed from a promoter not offered with the Flexi® Vectors. Here we discuss two strategies for transferring the HaloTag® coding region from Flexi® Expression Vectors into non-Flexi® expression Vectors.

By Shoba Ananthanarayanan, M.S., Michael Slater, Ph.D., and Trista Schagat, Ph.D.
Promega Corporation

Published in January 2009

Introduction

The HaloTag® Technology is a versatile fusion protein platform that allows biochemical and cell biological analyses with a single fusion construct (1). The HaloTag® coding sequence and optimized linker are available in the Flexi® Vectors in both N- and C-terminal orientations. The Flexi® Vector System is a rapid, efficient directional cloning method for protein-coding sequences (2). The pFN and pFC (HaloTag® 7) Flexi® Vectors are available for mammalian, E. coli and cell-free protein expression via the CMV, T7, and SP6 promoters (3,4).

Expression in other systems such as insect or yeast requires transfer of the HaloTag® coding region from the Flexi® Vector to an expression vector with the necessary regulatory elements (e.g., actin 5C promoter for insect cells or ADH1 promoter for yeast). This can be done in two ways:

  1. Create the full-fusion construct in the Flexi® Vector, then transfer the protein fusion to a different expression vector, or
  2. Transfer the HaloTag® coding region from the Flexi® Vector into a different expression vector and create the fusion construct after transfer.

Strategy# 1, creating the full fusion in the Flexi® Vector prior to transfer, is recommended because the Flexi® Vector cloning strategy makes it simple to clone the open reading frame of your protein of interest in-frame with the HaloTag® sequence and its optimized linker with minimal addition of extraneous amino acids.

Strategy# 1: Create a HaloTag® Fusion in the Flexi® Vector, Then Transfer

The Flexi® Vector cloning strategy makes it simple to clone the open reading frame (ORF) of your protein of interest in-frame with the HaloTag® coding region. Once this cloning step is complete, the HaloTag® fusion construct can then be subcloned from the Flexi® Vector to a second, non-Flexi® expression vector. The basic steps are as follows:

  1. Choose a Flexi® Vector containing the HaloTag® coding region. The pFN21K (HaloTag® 7) CMV Flexi® Vector (Cat.# G2831) or pFC17A (HaloTag® 7) CMVd3 Flexi® Vector (Cat.# G1551) can be used to create an N- or C-terminal HaloTag® construct, respectively. (For discussion on N- versus C-terminal fusions, see reference 3.)
  2. Amplify the ORF using PCR primers designed with unique Flexi® Vector restriction sites. (Primer design is simplified using the Promega Flexi® Primer Design Tool.)
  3. Subclone the ORF PCR product into the Flexi® Vector by digesting with restriction enzymes and ligating the vector with insert per the "Cloning PCR Products into the Flexi® Vectors (Entry Reaction)" section of the Flexi® Vector Systems Technical Manual #TM254. Confirm the ORF by sequencing analysis.
  4. Subclone the HaloTag® fusion construct from the Flexi® Vector to a non-Flexi® expression vector using unique restriction sites. Not all HaloTag® coding region-containing Flexi® Vectors have the same unique restriction sites so the subcloning strategy will be based on the vector used.

The pFN21K and pFC17A Flexi® Vector sequences were run through a web-based restriction enzyme site finder to look for unique sites. Single cutters adjacent to the HaloTag® ORF fusion site are recommended (Figure 1). Several two- or three-site cutters were also carefully selected to avoid enzymes that cut within HaloTag® or the linker sequence. It is critical to check which enzymes cut the ORF before choosing a specific restriction enzyme for transfer of the HaloTag® protein fusion.

thumbnail-Restriction sites for subcloning HaloTag® fusion constructs from the Flexi® pFN21K (Panel A) and pFC17A (Panel B) Flexi® Vectors.
Restriction sites for subcloning HaloTag® fusion constructs from the Flexi® pFN21K (Panel A) and pFC17A (Panel B) Flexi® Vectors.

Figure 1. Restriction sites for subcloning HaloTag® fusion constructs from the Flexi® pFN21K (Panel A) and pFC17A (Panel B) Flexi® Vectors. Following Strategy# 1, a fusion between the HaloTag® coding region and the ORF of your protein is created in a Flexi® Vector containing the HaloTag® coding region. These vectors contain optimized linker sequences with TEV protease cleavage sites (TEV). Unique restriction sites flanking the fusion construct are then used to subclone the fusion protein sequence into a non-Flexi® vector.
*Restriction enzymes that cut the vector more than once but outside the HaloTag® region.
**PmeI is a unique site but cuts at the AA/T stop codon. If this enzyme is chosen, then the stop codon must be recreated or present in the non-Flexi® vector.

In the absence of a suitable site, site-directed mutagenesis can be used to introduce the desired restriction endonuclease site. Several commercial kits exist to perform this, including the Promega GeneEditor™ Site-Directed Mutagenesis System (Cat.# Q9280) and the Stratagene QuikChange® Site-Directed Mutagenesis Kit. Alternatively, overhangs left by restriction endonucleases can be shortened or converted to blunt ends using DNA Polymerase I Large (Klenow) Fragment (Cat.# M2201) or T4 DNA Polymerase (Cat.# M4211; see the Cloning chapter of the Protocols and Applications Guide for protocol information.)

Strategy# 2: Transfer HaloTag® Coding Region to a Non-Flexi® Vector, Then Create a Fusion

In cases where transfer of the complete HaloTag® fusion construct is not desirable, the HaloTag® coding sequence can be transferred from the Flexi® Vector first, and then the fusion construct created in the non-Flexi® Vector. We recommend that the HaloTag® coding sequence and the optimized linker region carrying the TEV protease cleavage site both be transferred from the Flexi® Vector. The basic steps are below:

  1. Choose a Flexi® Vector containing the HaloTag® coding region. The pFN21K (HaloTag® 7) CMV Flexi® Vector (Cat.# G2831) or pFC17A (HaloTag® 7) CMVd3 Flexi® Vector (Cat.# G1551) can be used to create an N- or C-terminus fusion of HaloTag® 7, respectively. (For discussion on N- versus C-terminal fusions, see reference 3.)
  2. Subclone the HaloTag® coding region and linker coding sequence from the Flexi® Vector to a non-Flexi® expression vector using unique restriction sites (Figure 2).
  3. Subclone the ORF (containing start and stop codons as needed) into the non-Flexi® expression Vector in-frame with the HaloTag® coding region. If a PCR product is used, confirm the ORF by sequencing.
thumbnail-Restriction sites for subcloning HaloTag® from the pFN21K (Panel A) and pFC17A (Panel B) Flexi® Vectors.
Restriction sites for subcloning HaloTag® from the pFN21K (Panel A) and pFC17A (Panel B) Flexi® Vectors.

Figure 2. Restriction sites for subcloning the HaloTag® coding region from the pFN21K (Panel A) and pFC17A (Panel B) Flexi® Vectors. Following Strategy# 2, unique restriction sites flanking the coding sequence are used to subclone the HaloTag® coding region and linker (TEV) sequence from a Flexi® Vector into a non-Flexi® vector. A fusion with the ORF of the protein of interest is then created.
*NcoI cuts the pFN21K Flexi® Vector three times. The 939bp HaloTag®-containing fragment must be gel purified to remove the 3.5kb and 552bp fragments, and constructs must be checked for insert orientation.

The disadvantage of this strategy is the potential challenge of cloning the HaloTag® coding region in-frame with the ORF in the non-Flexi® Vector while not adding extraneous amino acids that could affect protein function. This requires careful sequence analysis and choice of cloning sites that will minimize the distance between the TEV linker and the ORF.

Summary

The Flexi® Vectors are available for expression of N- and C-terminal HaloTag® fusion proteins in mammalian cells, E. coli, and in vitro via CMV, T7 and SP6 promoters. If different promoters are needed for expression in other systems such as yeast and insect cells, the HaloTag® sequence can be transferred from a Flexi® Vector to an appropriate expression vector. For easy in-frame cloning and minimal addition of extraneous amino acids, we recommend first cloning the ORF into a Flexi® Vector containing HaloTag® coding region, then transferring the complete fusion construct to the non-Flexi® expression vector.

References

  1. Los, G.V. et al. (2005) HaloTag™ Interchangeable Labeling Technology for cell imaging, protein capture and immobilization. Promega Notes 89, 2–6.
  2. Slater, M. et al. (2005) A new system for cloning and expressing protein-coding regions. Promega Notes 89, 11–15.
  3. McCornack, M., Schagat, T. and Slater, M. (2008) Expression of fusion proteins: How to get started with the HaloTag® Technology. Promega Notes 100, 13–15.
  4. Slater, M. et al. (2008) Achieve the protein expression level you need with the mammalian HaloTag® 7 Flexi® Vectors. Promega Notes 100, 16–18.