Construction of a Gene Fusion of Streptavidin with a Partner Protein

Expression vectors have been designed that are useful for the construction of a gene fusion of streptavidin with a partner protein (Fig. 1) (10). These fusion vectors, pTSA-18F and pTS A-19F, carry a truncated streptavidin gene, encoding only the essential region of streptavidin (amino acid residues 16-133), with a translation initiation codon (ATG) under the control of the 010 promoter of bacteriophage TO, followed by the TO transcription terminator (7) A polylinker region, derived from pUC19, is placed downstream of the coding sequence for streptavidin, so that a gene fusion encoding a chimera consisting of the N-terminal streptavidin moiety and the C-terminal partner protein moiety can be easily constructed. Alternatively, the 3'-terminus of a coding sequence for a partner protein can also be fused to the 5'-terminus of the truncated streptavidin gene to produce a chimeric protein, consisting of the N-terminal partner protein moiety and the C-terminal streptavidin moiety, by inserting the partner protein gene in frame into a unique Ndel site located at the translation initiation site.

bla bla

pTSA-18F 2.7 kb pTSA-19F

Streptavidin (amino acids 16-133) Polylinker t<p

Streptavidin (amino acids 16-133) Polylinker t<p

Streptavidin gene EcoIf I,Sac 1 KP,n 1

SmaIXmaI BamH I

-aag gtg aat tcg agc tcg gta ccc ggg gat cct

Lys Val Asn Ser Ser Ser Val Pro Gly Asp Pro

Streptavidin gene EcoIf I,Sac 1 KP,n 1

SmaIXmaI BamH I

-aag gtg aat tcg agc tcg gta ccc ggg gat cct

Lys Val Asn Ser Ser Ser Val Pro Gly Asp Pro

Acc I Sal I Sph I Hind III

Fig. 1. Expression vectors, pTSA-18F and pTSA-19F, for streptavidin-containing chimeric proteins. These vectors carry a coding sequence for only the essential region of streptavidin (amino acid residues 16-133) with a translation initiation codon (ATG) under the control of the &10 promoter of bacteriophage T7 A polylinker region, derived from pUC19, is placed downstream of the coding sequence. This polylinker region greatly facilitates the construction of gene fusions of streptavidin with partner proteins. (Reproduced with permission from ref. 10, copyright, Academic, Orlando, FL, 1991 )

Standard molecular cloning methods and procedures are used for construction of a gene fusion of streptavidin with a partner protein (8,9). The resulting expression veci or should be stably maintained in E coli strains commonly used for cloning, such as DH5a, unless they carry the T7 RNA polymerase gene or its fragment.

The expression vector encoding a streptavidin-partner protein chimera is isolated from host E. coli cells by using standard plasmid minipreparation methods (8,9). The isolated expression vector is used to transform E coli lysogens, BL21(DE3)(pLysS) and BL21(DE3)(pLysE), which carry the T7

RNA polymerase gene under the /acUV5 promoter in the chromosome. These lysogen strains also contain an additional plasmid, pLysS or pLysE, which carries the T7 lysozyme gene under the tet promoter and the chloramphenicol acetyltransferase (CAT) gene. Constitutive production of T7 lysozyme, which is a natural inhibitor for T7 RNA polymerase and is used to reduce the basal T7 RNA polymerase activity in an uninduced state (7), is essential to maintain toxic genes, such as the streptavidm gene, stably in host BL21(DE3) lysogens.

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