Optimizing the expression of a Heterologous chitinase: A study of different promoters

Figures & data

Figure 1. SDS-PAGE showing the expression of the ChiA1-2 gene using BL21ChiA1-2Ll1 (A) and BL21ChiA1-2Ll2 (B) strains induced with IPTG. Lane 1: molecular weight protein marker (A: Hide Range Sigma Marker™, Sigma-Aldrich; B: Protein Marker, Broad Range, Biolabs); lane 2: cell extract before induction; lanes 3–4: cell extract 2 and 4 h, respectively, after induction. The recombinant protein fused with the GST tag is boxed.

Table 1. Effect of the promoters and different induction conditions on the expression of the active form of the chitinase encoded by the ChiA1-2 gene.

Activity (U/mL)
Parameter analyzed	BL21ChiA1-2Ll1^a	BL21ChiA1-2Ll2^b
OD600 nm
0.5	240.7 ± 0.4	38.4 ± 2.7
0.8	98.7 ±2 .4	55.8 ± 1.1
1.2	95.1 ± 0.8	66.3 ± 4
IPTG concentration (mM)
0.12	89.4 ± 5	62 ± 4.3
0.25	108 ± 0.4	56.8 ± 1.2
0.5	98.7 ± 1.5	55.8 ± 1.1
Post-induction time (h)
1	152.7 ± 6	45.2 ± 0.9
5	98.6 ± 0.5	55.8 ± 2.8
24	66.2 ± 5.2	33 ± 0.4

^a Expressing the gene from the tac promoter.

^b Expressing the gene from the T7 promoter.

Table 2. Strains and plasmids used in this study.

Strain	Source	Genotype/characteristics
L. lactis sp. lactis CECT 185	CECT^a	Wild type
E. coli One Shot® TOP10	Invitrogen	F^− mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15ΔlacX74 recA1 araD139Δ(ara-leu)7697 gal/U galK rpsL (Str^R) endA1 nupG
E. coli BL21 (DE3)	Invitrogen	F^− ompT gal dcm lon hsdSB(rB^− mB^−) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5])
E. coli BL21 Star (DE3)	Invitrogen	F^− ompT hsdSB (rB^− mB^−) gal dcm rne131 (DE3)
BL21ChiA1-2Ll1	This study	E. coli BL21 (DE3) transformed with pGEX-4T-2-ChiA1-2Llp recombinant vector
BL21ChiA1-2Ll2	This study	E. coli BL21 Star (DE3) transformed with pET41-ChiA1-2Llp recombinant vector
Plasmid
pCR®- Blunt	Invitrogen	Cloning vector. Confers zeocin and kanamycin resistance
pGEX4T-2	Amersham	IPTG inducible expression vector under control of the tac promoter. Introduces a GST tag at the N-terminus of the expression protein. Confers ampicillin resistance
pET41 EK/LIC	Novagen	IPTG inducible expression vector under control of the T7 promoter. Introduces a GST tag at the N-terminus of the expression protein. Confers kanamycin resistance
pTOPChiA1-2Llp	This study	pCR®- Blunt vector containing the ChiA1-2 gene
pGEX-4T-2-ChiA1-2Llp	This study	pGEX4T-2 vector containing the ChiA1-2 gene.
pET41-ChiA1-2Llp	This study	pET41 EK/LIC vector containing the ChiA1-2 gene.

^a CECT: Spanish Type Culture Collection.