Of a Streptavidin Partner Protein Chimera in E coli

Expression of a gene fusion encoding a streptavidin-partner protein chimera is carried out by using the T7 expression system (7), which allows efficient production of various streptavidm mutants and streptavidin-containmg chimeras. The procedure described below is written for a culture volume of 100 mL and can be scaled up as needed.

1. BL21(DE3)(pLysS) or BL21(DE3)(plLysE) carrying an expression vector encoding a streptavidin-partner protein chimera is grown overnight at 37°C with vigorous shaking m 5 mL of M9 minimal medium supplemented with 1 mM MgS04, 0.2% glucose, 1.5 \iM thiamine, 0.5% Casamino acids, 8 2 pMbiotin, 150 pg/mL ampicilhn, and 25 pg/mL chloramphenicol.

2 Approximately 2 mL of fresh culture of BL21(DE3)(pLysS) or BL21(DE3) (pLysE) carrying the expression vector is added to 100 mL of M9 minimal medium supplemented with 1 mMMgS04,0 2% glucose, 1 5 pMthiamine, 0 5% Casamino acids, 8 2 pMbiotin, 150 pg/mL ampicilhn, and 25 pg/mL chloramphenicol. Cells are grown at 37°C with vigorous shaking

3 When the absorbance of the culture reaches 1.0 for cells carrying pLysS or 0 6 for those carrying pLysE, 100 mM isopropyl-p-d-thiogalactopyranoside (IPTG) is added to a final concentration of 0 4—0.5 mM to induce the expression of the T7 RNA polymerase gene placed under the /acUV5 promoter. After induction, cells are incubated at 37°C with vigorous shaking

Expression of the encoded streptavidin-partner protein is analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) (11) of total cell protein (see Fig. 2). A part of the culture (e.g., 500 pL) is collected periodically m a microcentrifuge tube, and cells are precipitated by brief cen-trifugation. The supernatant is discarded, and the cell pellet is dissolved in a sample solution for SDS-PAGE, such as 3% SDS, 100 mMTns-Cl (pH 6.8), 30% glycerol, 0.01% bromophenol blue. The dissolved sample is heated in boiling water for 3 min, and applied to a polyacrylamide gel of an appropriate polyacrylamide concentration. Total cell protein from approx 100 fiL of culture is applied to each lane, but appropriate amounts need to be found empiri-

124 Sano, Smith, and Cantor

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Fig. 2. Expression of a streptavidin-metallothionein chimera in E. coli Total cell protein of E. coli BL2l(DE3){pLysE) carrying an expression vector, p'TSAMT-2, encoding a streptavidin-metallothionein chimera was analyzed by SDS-PAGE using a 15% Polyacrylamide gel. Proteins were stained with Coomassie brilliant blue R-250. Lanes a, BL2l(DE3)(pLysE) without the expression vector; b, BL2l(DE3){pLysE) (pTSAMT-2). The expressed streptavidin-metallothionein chimera is indicated by an arrow. The number above each lane is the time in hours after induction. Each lane contains the total cell protein from 167 pL of culture, except for the lane at 5 h for lanes a, where 83 pL of culture was used. (Reproduced with permission from ref. 14.)

cally according to the growth of host cells and gel electrophoresis apparatus used. Proteins are stained with Coomassie brilliant blue R-250. Usually, expressed chimera is the dominant species in total cell protein and thus can be easily identified.

Generally, expression of a streptavidin-partner protein chimera reaches a maximum at 2-3 h after induction for BL21 (DE3)(pLysS) and at around 4 h after induction for the equivalent strain carrying pLysE. Expression efficiencies arc generally higher with BL21(DE3)(pLysE) than with BL21(DE3) (pLysS). However, degradation of expressed chimera by cellular proteinases is generally greater with BL21(DE3)(pLysE). Thus, for a particular streptavidin-partner protein chimera, both strains should be first tested to see which strain has higher expression efficiency and provides a higher yield of purified, functional chimera molecules

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