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Patrick C.H. Lo, Ph.D. Kristie Nybo, Ph.D.
BioTechniques, Vol. 46, No. 6, May 2009, p. 391
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Curing the blas

The necessity for structural biology laboratories to clone and express a large number of different proteins in a timely and efficient manner highlights the need to quickly identify the correct clones of numerous protein expression constructs. Existing vectors for the positive selection of clones with an insert are only suitable for cloning and not the direct high-level expression of proteins, and many cannot select for the correct insert orientation. In this issue, A. Haag and C. Ostermeier at the Novartis Institutes for BioMedical Research (Basel, Switzerland) present their protein expression vector for the positive selection of clones with inserts in the proper orientation that can be used directly for high-level protein expression. Their positive-selection strategy is based on the observation that the C-terminal–most amino acid of the β-lactamase (bla) gene for ampicillin resistance is a tryptophan (W290) that is essential for full function and cannot be deleted or changed to any other amino acid. Their vector contains a truncated bla gene missing the W290 residue. If the protein-coding sequence to be cloned is PCR-amplified with a 3′ primer containing a tryptophan residue at its very 3′ end in the opposite orientation to the protein sequence, then the insertion of this insert tail-to-tail with the truncated bla gene will reconstitute its missing W290 residue, thus restoring its function and allowing for selection by ampicillin resistance of clones containing the insert in the correct orientation for expression. The insert is expressed from a T7 polymerase–based expression cassette, and there is no fusion protein with β-lactamase since the two genes are transcribed independently tail-to-tail and are separated from each other by tandem stop codons. They tested the vector using gfp and mCherry as target inserts and demonstrated that there were no false positives in >20 experiments with 4–16 colonies examined, and that the high-level expression of the insert proteins was not affected by the truncated bla selection cassette.

(See “Positive-selection vector for direct protein expression”.)

HIV-1 Cloning: Pieced in Yeast

Virus isolation and propagation is challenging for many species and strains, so researchers often clone specific virus genes into vectors for functional studies instead. Highly heterogeneous viruses such as human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) lack unique conserved restriction enzyme sites, complicating their use in cloning experiments. As a result, analyses of HIV-1 have been conducted on only a few available subtype B laboratory strains that do not accurately represent the HIV-1 isolates from the infected population (which can differ by as much as 25% in nucleotide sequence). One alternative to bacterial cloning is the homologous recombination in a human cell line of a proviral DNA vector containing a deletion in a specific gene with the corresponding gene PCR-amplified from an HIV-1 infected patient sample. This results in a full-length HIV-1 genome and infectious virus, but is also inefficient and biased towards a few randomly generated clones. To overcome these problems, D. Dudley and colleagues in the laboratory of E. Arts at Case Western Reserve University (Cleveland, OH) had previously developed a version of the method using yeast-based homologous recombination, which is more efficient and precise. Now, in this issue, they demonstrate a greatly improved version that dispenses with standard subcloning steps and allows any HIV-1 coding region PCR-amplified from a clinical sample of any subtype to be easily and efficiently cloned into an appropriate, nearly full-length HIV-1 DNA shuttle vector through a single cloning step in yeast involving negative and positive selection markers to ensure insertion of the desired sequence. In order to prevent yeast-mediated recombination of the LTRs that would delete the entire coding region, the 5′ LTR of the HIV-1 genome in the shuttle vectors was removed. Transfection of a shuttle clone into a modified 293T packaging cell line providing the RNA sequence of the HIV-1 LTR in trans generates replication-competent virus with dipartite genomes, which are able to infect a producer cell line, causing robust production of virus with full-length chimeric HIV-1 genomes. The cloning strategy is also being developed for HCV and influenza virus.

(See “A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates”.)