English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Zendy Open

Presents the pdf analysis as premium feature

PDF Analysis

Insights

Presents the summarisation as premium feature

Summarisation

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the zaia usage as premium feature

ZAIA

Zendy Tools

Zendy Plus

Presents the access of premium content as premium feature

Premium Content

Blue/white color selection, based on insertional inactivation of /3-galactosidase (1), is a powerful tool for DNA cloning in E. coli. However, proteins expressed from such recombinants are fusion proteins. Although these are very valuable, non-fused proteins are much more desirable for many biochemical experiments. We describe here a general method for expression of non-fused proteins which utilizes blue/white selection. Using PCR (2), the 5' non-coding region of a full length Trypanosoma brucei glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) cDNA (3) was replaced by prokaryotic signals for translation initiation and enhancement. The upstream PCR primer (Figure 1A) contains a BamHl site (GGATCC), an appropriately spaced putative translational promoter (GATCC) for highly expressed E. coli proteins (4), the X cro Shine —Dalgarno sequence (AGGAGG), a modified lacZ spacer region (CAGCTAA), and 12 nucleotides (bold) of the GPI-PLC coding sequence; there are also translational termination codons (TAA; outlined) to block synthesis of any protein encoded upstream of the GPI-PLC gene. The downstream primer (Figure 1A) contains 21 nucleotides complementary to the 3' end of GPI-PLC coding sequence (bold), a //mdllll site, Mrtdllll cleavage. Functional capacity of the regulatory elements in the upstream primer was established when GPI-PLC encoded by a similar PCR product was expressed in E. coli, under control of the X PL promoter, after cloning into pRLM76 (from J.D.Roberts and R.McMacken, a derivative of pHE6 (5)). Data available on request. After PCR for 25 cycles the product was digested with BamHl and Hindlll and cloned into pBluescript SK + / (Stratagene). The resulting construct, shown in part in Figure IB, was transformed into E. coli DH5a. Transcription from the 0galactosidase promoter (Figure IB) should give rise to a dicistronic mRNA encoding two distinct proteins: one is a shorter form of the a fragment of /?-galactosidase, truncated by an inframe termination codon introduced by the upstream PCR primer (see Figure 1); and the other is full length GPI-PLC. White clones in a background of blue were selected, grown and induced with IPTG (1). After SDS-PAGE of all lysates (5) GPI-PLC was detected by Western blotting (6). A 39 kDa immunoreactive protein was produced, e.g. in PGPIPLC-08, which was identical in mobility to that of the T. brucei enzyme and to the product in cells containing pRLM76-36. A fusion protein containing j3galactosidase sequences (predicted 43 kDa) was not detected. With a similar constuct we have produced enzymatically active GPI-PLC (in preparation). This cloning method should be adaptable for expression of other authentic (non-fusion) proteins in E.coli.

Non-fusion proteins expressed inE.coli: detection by α-complementation (blue/white selection