The expression vectors

Answer C. Incorporation of a Shine-Dalgarno sequence into the expression vector will promote ribosome binding to the translation start site on the mRNA produced by transcription of the cDNA insert. 

The master cell bank is a homogeneous suspension of the original cells already transformed by the expression vector containing the desired gene, distributed in equal volumes into individual containers for storage (e.g. in liquid nitrogen). In some cases it may be necessary to establish separate master cell banks for the expression vector and the host cells. 

Fig. 1. The expression vector pTDI. (a) pTDI vector map. (b) DMA sequence of the pTDI vector around the multiple cloning sites.

The pTDl vector should not be used as the expression vector for an E. coli cell-free protein synthesis system because this vector does not contain Shine-Dalgarno sequence. 

During this process some key questions are (1) Does the DNA sequence inserted into the expression vector contain all the sequences essential for the target protein (2) Is the messenger RNA expressed at the predicted length and produced in high enough levels (3) Does the recombinant protein react with antibody (4) Is there evidence of functional activity of the recombinant protein Methods for answering these questions are listed in Table 4.4. 

Preliminary site-directed mutagenesis experiments of the fused 1,2-a-D-mannosidase gene (f-msdS) on the expression vector, pGAM-1, were done previously to identify the functional role of catalytic residues in the 1,2-a-D-mannosidase from A. saitoi expressed in yeast cells [113],... 

The success of transfection, either stable or transient, depends on several factors that must be taken into account and can be summarized as follows (i) the transfectability and physiology of the cell line (ii) the characteristic of the genetic marker in the expression vector (iii) the type of expression desired (iv) the size of the expression cassette and the quality of the DNA to be introduced (v) the compatibility of the transfection method and/or reagents with the cell line (vi) the type of assay to be used for detection of... 

In the next sections, the transfection methods most commonly used in cell culture laboratories will be described. It is difficult to predict the best method for transfection, so the expression vector, the cell type, and the facilities of each laboratory should be taken into account before deciding which technique to apply (Wurm, 2004). For infection techniques, an updated compilation can be obtained from Heiser (2004). 

Figure 10.12 Gas chromatograms of the reaction products formed upon incubation of microsomes isolated from yeast over-expressing a tobacco terpene hydroxylase gene (panels A C) (CYP71D20) or harboring only the expression vector DNA (control) (B D). Microsomes were incubated with 5-epi-aristolochene (A B) or 1-deoxycapsidiol (C D) in the presence (blue line) or absence (red line) of NADPH. 5-epi-aristolochene, 1-deoxycapsidiol and capsidiol were all verified by MS.58...

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