Enzyme and Microbial Technology, Vol.40, No.1, 85-92, 2006
Production of a recombinant form of the propeptide NH2-terminal of the precursor of pulmonary surfactant protein B
The human surfactant protein B (SP-B) is an small protein produced by the proteolytic processing of a precursor through elimination of two propeptides flanking the mature protein at its NH2- and COOH-termini, respectively. The human N-terminal propeptide (SP-B-N) sequence has been cloned into the expression vector pMAL-c2x fusioned to maltose-binding protein. Expression of the fusion protein MBP-SP-B-N has been achieved in Escherichia coli UT5600 cells after IPTG induction. The fusion protein was purified by affinity chromatography through an amylose resin column and identified by Western blot analysis with anti-MBP and anti-proSP-B antibodies. Proteolytic cleavage with Factor Xa followed by anion exchange chromatography allowed separation of the propeptide SP-BN from MBP. Mass spectrometry gave a molecular mass of 19 883 Da for the propeptide and its tryptic digestion produced peptides covering partly the propeptide sequence. An amount of 0.18 mg of propeptide per L culture was obtained. (c) 2006 Elsevier Inc. All rights reserved.