Expression by streptomyces lividans of the rat alpha integrin CD11b A-domain as a secreted and soluble recombinant protein

Abstract

We already reported the use of a long synthetic signal peptide (LSSP) to secrete the Streptomyces sp. TO1 amylase by Streptomyces lividans strain. We herein report the expression and secretion of the rat CD11b A-domain using the same LSSP and S. lividans as host strain. We have used the Escherichia coli/Streptomyces shuttle vector pIJ699 for the cloning of the A-domain DNA sequence downstream of LSSP and under the control of the constitutive ermE-up promoter of Streptomyces erythraeus. Using this construct and S. lividans as a host strain, we achieved the expression of 8 mg/L of soluble secreted recombinant form of the A-domain of the rat leukocyte beta2 integrin CD11/CD18 alpha M subunit (CD11b). This secreted recombinant CD11b A-domain reacted with a function blocking antibody showing that this protein is properly folded and probably functional. These data support the capability of Streptomyces to produce heterologous recombinant proteins as soluble secreted form using the "LSSP" synthetic signal peptide.

Figure 1

Construction of plasmid pMS115. The constitutive ermE-up promoter from pIJ4070 was subcloned as a SacI-BamHI fragment in the pMS39 plasmid carrying the long synthetic signal peptide (LSSP) giving the pMS41 plasmid. Then the KpnI-HincII fragment carrying PermE-up-LSSP from pMS41 was cloned in pIJ4070 yielding the pMS51 plasmid. PCR product of the rat CD11b A-domain was cloned into the pCR-Blunt vector to produce the pMS52 plasmid. The fragment EcoRV-PstI corresponding to the A-domain from pMS52 was cloned in pMS51 giving the pMS62 plasmid. Finally, the BglII fragment from pMS62 carrying the cassette “PermE-up-LSSP-A-domain” was cloned in the pIJ699 vector yielding the pMS115 plasmid.

Figure 2

RT-PCR analysis of the A-domain expression in S. lividans 1326/pMS115. Lane 1: smart ladder molecular weight (MW) marker (Eurogentec). Lanes 2, 3, and 4: RT-PCR performed with cDNA synthesised from 2 μg of RNA treated with DNase and prepared from the S. lividans 1326/pMS115 strain grown on TSB medium for 36 h, 50 h, and 60 h, respectively. Lane 5: PCR performed directly with 2 μg of RNA treated with DNase (control).

Figure 3

Western blot analysis of the A-domain secretion in S. lividans 1326/pMS115. Supernatant of the S. lividans 1326/pMS115 strain grown on NMMP medium was concentrated 50x, lyophilized, resuspended in PBS, then dialyzed as described in “Materials and Methods.” Proteins were run on a 15% SDS polyacrylamide gel. Lane 1: molecular weight standards (MW) from Amersham; lane 2: 15 μg of supernatant proteins from S. lividans 1326/pMS115; lane 3: 2 μg of the recombinant A-domain (21 kDa) released from the GST/CD11b A-domain fusion protein (48.5 kDa) by thrombin cut; lane 4: 2 μg of the GST/CD11b A-domain fusion protein (48.5 kDa).