Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion.

Yue Wu,Jiannan Wang,Zhifang Tian,Zhao Kang,Mingyang Wu

doi:10.3390/polym9120669

Abstract

In order to investigate the impacts on the structure and biomedical function of typical fragments derived from repetitive and non-repetitive regions of the Bombyx mori silk fibroin heavy chain, several block combination genes (gs16f1, gs16f4, gs16f8, and gs16f12) were designed, cloned into a fusion protein expression vector tagged with glutathione S-transferase (GST), and expressed in Escherichia coli. Fusion proteins GST-GS16F1, GST-GS16F4, and GST-GS16F8 were purified by GST affinity chromatography, and single bands were identified by SDS-PAGE. Under optimal initial cell density, in ducer concentration and induction expression time, the yield of purified GST-GS16F1, GST-GS16F4, and GST-GS16F8 per liter of bacterial culture reached 79, 53, and 28 mg, respectively. Mass spectrometry revealed molecular weights for GST-GS16F1, GST-GS16F4, and GST-GS16F8 of 37.7, 50.0, and 65.7 kDa, respectively, consistent with the theoretical values of 37.4, 49.4, and 65.5 kDa. Similarly, measured values of pI were 5.35, 4.5, and 4.2 for the fusion proteins, consistent with predicted values of 5.34, 4.44, and 4.09. CD spectra showed the molecular conformation of GS16F1 was mainly β-sheet structure, while more stable α-helix structure formed in GS16F4 and GS16F8.

Highlights

Bombyx mori (B. mori, Lepidoptera) produces silk fibroin that is linked to biomedical applications owing to its high biocompatibility, degradability and ease of fabrication [1,2,3,4,5,6]
The gs16f1 gene encoding a structural component of H-Chain was cloned, and recombinant
The gs16f1 gene encoding a structural component of H-Chain was cloned, and recombinant gs16f1, gs16f1, gs16f4, and gs16f8 containing different numbers of the components were stably expressed as gs16f4, and gs16f8 containing different numbers of the components were stably expressed as glutathione S-transferase (GST) fusion

Summary

Introduction

Bombyx mori (B. mori, Lepidoptera) produces silk fibroin that is linked to biomedical applications owing to its high biocompatibility, degradability and ease of fabrication [1,2,3,4,5,6]. H-Chain is the major component and includes an N-terminal region, a major core region, and a C-terminal region. The core domain consists of 12 repetitive domains and 11 non-repetitive fragments [7], and this portion can be used as a model structure to investigate the relationship between structure and function, including cell functions. Numerous reports have discussed the relationship between the sequence, conformation and characteristics of silk fibroin analogues based on chemical synthesis and genetic engineering methods. The (GAGAGS) hexapeptide is the core unit of H-Chain and plays an important role in the formation of crystalline domains [12,13]. Silk protein-like multiblock polymers derived from the repetitive domain of B. mori silk and spider dragline silk spontaneously aggregate into β-sheet structures, similar to natural silks [14,15,16].

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Conclusion