The expression, purification and characterization of recombinant human lung surfactant protein-B

Abstract

A way to produce surfactant protein B (SP-B) in the laboratory is highly sought both for basic research on SP-B, as well as for use as a component of surfactant for treatment of patients in respiratory distress. Herein, I hypothesize that SP-B can be expressed in bacteria in its functional form. In the long run, I also anticipate that recombinant SP-B could alleviate the financial burden incurred by the health care system during the treatment of neonatal respiratory distress syndrome patients. Moreover, I envisage, recombinant surfactant protein B variants with an improved capability to resist surfactant dysfunction. In the first part of the study, recombinant DNA technology was used to overexpress the near full-length human SP-B variant, Δ7NTΔM-SP-B- 6xHis, in BL21 Escherichia coli , strain C43 cells. The results show that recombinant surfactant protein B expresses as inclusion bodies but can be renatured carefully using immobilized metal ion affinity chromatography. Importantly, the thesis work has established a protocol for the production of recombinant surfactant protein B that is amenable to scale up. In the next part of the study, circular dichroism was used to assess the conformation of recombinant SP-B suspensions in different membrane mimetic environments. Likewise, dynamic light scattering was used to characterize sample homogeneity and aggregation propensity of the protein suspension in these conditions. It was found that dodecyl phosphocholine/sodium dodecyl sulfate binary micelles and methanol support native-like secondary structure of recombinant SP-B comparable to animal derived surfactant protein B. In the last portion of the study, the function of recombinant SP-B was tested in lipid environments using the Langmuir-Wilhelmey surface balance. The results indicate that recombinant SP-B possesses the necessary biophysical features to promote the large-scale organization of lipid monolayers that are thought to be critical to SP-B function. In conclusion this work supports the use of recombinant SP-B in a surfactant therapeutic as a potential future alternative to animal derived SP-B.