Directional cloning systems

To increase the amount of DNA covered in each step, three different strategies have been used One approach, the use of vector-host systems likely to allow the direct cloning of DNA fragments larger than those accommodated in cosmid, has led to the recent development of artificial yeast cloning vectors (13). 

Marchuk D, Drumm M, Saulino A, Collins FS (1990) Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res 19 1154... 

Directed evolution relies on the analysis of large numbers of clones to enable the discovery of rare variants with unproved function. In order to analyze these large libraries, methods of screening or selection have been developed, many of which use specialized equipment or automation. These range from the use of multichannel pipettes, all the way up to robotics, depending on the level of investment [59]. Specialized robotic systems are available to perform tasks such as colony picking, cell culture, protein purification, and cell-based assays. 

Cell lines, such as the Caco-2 and MDCK cells [27, 35, 47, 49, 57, 67, 128-133], have been used frequently to study different transporters in the GI tract. These cell lines have been evaluated for transport both in absorptive and secretory direction and in addition also been transfected with specified transporter systems of interests to yield new clones [23, 31, 72, 79, 80, 134] or co-cultures [135], Some of the uptake transporters belonging to the organic cation transporter (OCT) family have also been identified in cell lines such as the pig kidney cell line LLC-PK1, and MDCK [67, 136]. In fact, its presence in Caco-2 cells needs to be further elucidated as reports have shown both the absence and presence of transporters from this family of transporters [136-138],... 

The staphylokinase gene has been cloned in E. coli, as well as various other recombinant systems. The protein is expressed intracellularly in E. coli at high levels, representing 10-15 per cent of total cellular protein. It can be purified directly from the clarified cellular homogenate by a combination of ion-exchange and hydrophobic interaction chromatography. 

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