Improved soluble expression of the gene encoding amylolytic enzyme Amo45 by fusion with the mobile-loop-region of co-chaperonin GroES in Escherichia coli

Figures & data

Table I. Strains and plasmids used in this study.

Strain or plasmid	Relevant features	Reference
Plasmids
pGro7	araPBAD expression vector containing genes for GroES and GroEL, Cm^r	TaKaRa BIO INC.
pJOE5751.1	L-rhamnose-inducible expression vector containing His6-eGFP, Amp^r	Wegerer et al. (2008)
pJOE3075	L-rhamnose-inducible expression vector, Amp^r	Stumpp et al. (2000)
pLEI45.1	expression vector containing His6-amo45, Amp^r	This study
pLEI88.1	expression vector containing mobile-loop-eGFP, Amp^r	This study
pLEI90.1	expression vector containing mobile-loop-amo45, Amp^r	This study
Strain
E.coli K12 JM109	F´traD36 proA+ B+ lacIq Δ(lacZ)M15/Δ(lac-proAB) glnV44e14- gyrA96 recA1 relA1 endA1 thi hsdR17	Yanisch-Perron et al. (1985)
E.coli JM109/pLEI45.1	Expression strain for His6-Amo45	This study
E.coli JM109/pLEI45.1/pGro7	Expression strain for His6-Amo45 and GroEL-GroES	This study
E.coli JM109/pLEI90.1	Expression strain for mobile-loop-Amo45	This study
E.coli JM109/pLEI90.1/pGro7	Expression strain for mobile-loop-Amo45 and GroEL-GroES	This study

Table II. Primers used in this study.

Primer	Nucleotide sequences	Application
GH57-ECY-f	GAGGATCGCAAACAACTGCaartgytayyt	Screening
GH57-GWFF-r	GGTGCTCGCCGAAGaaraaccanwc	Screening
amo45-1bio	GGATCCAGAGGGAGTATGGTATGGTTG	Retrieval
amo45-3	TGGTGGCCATAGCTCCCCTTCCT	Retrieval
amo45-2bio	CCTGGAGCTGTCCTCACGGTGTGGAA	Retrieval
amo45-4	TGGAAAGATGGAGGGATCACTGTG	Retrieval
amo45-6-bio	TACACTCCCAAAGACATAGCCGTTACTG	Retrieval
amo45-8	CCTCATGTACCTTGCTGATGTGGAAA	Retrieval
amo45-10-bio	TCCTTGACGCAGAATGCTGATCCT	Retrieval
amo45-12	AACCGTGGAGGAGATTCTCA	Retrieval
Arb1	GGCCACGCGTCGACTAGTACNNNNNNNNNNGATAT	Retrieval
Arb2	GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC	Retrieval
Arb3	GGCCACGCGTCGACTAGTAC	Retrieval
S8481	TATGGAAGTTGAAACCAAATCTGCTGGTGGTATCGTTCTGACCGG TTCTGCTGCTGCGG	synthesis of mobile-loop
S8482	GATCCCGCAGCAGCAGAACCGGTCAGAACGATACCACCAGCAGATTT GGTTTCAACTTCCA	synthesis of mobile-loop
S7422	AAAAAATGATCAATAATATTTCACGGTCACTTTTA	PCR for amo45
S7423	AAAAAAAAGCTTTCATAGTGTGGC	PCR for amo45

Figure 1. Expression of His₆-amo45 with and without co-expression of groEL–groES, analyzed by SDS-PAGE. Lanes 1–4: Crude extract of JM109 pLEI45.1 (His₆-amo45). Lanes 5–10: Crude extract of JM109 pLEI45.1/pGro7. The −, ++ and +/+ indicate uninduced, with arabinose and rhamnose simultaneously induced (4.5 h), and successively induced (3 h + 4.5 h) cell cultures, respectively. The M, S, and I labeled the molecular weight marker, soluble, and insoluble fractions, respectively.

Figure 2. Expression of mobile-loop-amo45 with and without co-expression of groEL-groES, analyzed by SDS-PAGE. Lanes 1–4: Crude extract of JM109 pLEI90.1 (mobile-loop-amo45). Lanes 5–10: Crude extract of JM109 pLEI90.1 pGro7. The −, ++ and +/+ indicate uninduced, with arabinose and rhamnose simultaneously induced (4.5 h), and successively induced (3 h + 4.5 h) cell cultures, respectively. The M, S, and I labeled the molecular weight marker, soluble and insoluble fractions, respectively.

Figure 3. Average specific enzyme activity of soluble crude extract samples containing His₆-Amo45 or mobile-loop-Amo45 measured by using synthetic substrate CNP-G3. The black, white, and gray columns present the specific activity of uninduced, with arabinose and rhamnose simultaneously induced (4.5 h), and successively induced (3 h + 4.5 h) cell cultures, respectively.

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