Exploring optimal expression conditions for low-degraded and low chaperone-contaminated recombinant silk protein

Abstract

Spider silk is a highly attractive biomaterial with exceptional strength and versatility, offering
potential applications across numerous industries. However, large-scale production remains a
significant challenge. Harvesting silk directly from spiders has proven impractical, making
recombinant protein production a promising alternative. Spider silk has been studied at Lund
University for several years, and this master’s thesis builds on previous work to evaluate four
constructs of a shortened spider silk gene and identify the most suitable construct for further
research. The host organism used for recombinant expression was the Escherichia coli strain
Turner (DE3).

The four His-tagged constructs were prepared prior to the start of the project. They were
cultivated, harvested, processed, and purified using immobilized metal affinity
chromatography (IMAC). Samples collected throughout the workflow were analysed by
SDS-PAGE to enable comparison of construct expression and purification efficiency.

No single construct could be identified as superior. Detectable bands corresponding to the
purified protein were difficult to obtain on SDS-PAGE, and the results were insufficient to
determine an optimal construct. Extensive optimization of the IMAC protocol was
performed; however, improvements and modifications targeting the insoluble fraction did not
yield consistent results. These limitations suggest that the underlying issue may be low
expression levels or instability of the constructs, which complicates reliable detection.

Popular Abstract

Producing Spider Silk with the Help of Bacteria

Imagine being able to produce a super-material in a laboratory flask, with the help of bacteria
instead of spiders. Spider silk without handling spiders sounds like science fiction. It is a
dream that is now within reach, but not quite yet fully realized.

In this project, we succeeded in producing spider silk proteins using bacteria. However,
producing enough of the material to collect and purify it remains a major challenge.

Spider silk has fascinated scientists for decades. How can something so thin be so strong? It
is often described as a “super-material” because of its extraordinary strength combined with
impressive elasticity. It is sometimes even compared to Kevlar. Because of these properties,
spider silk has been proposed for many uses: from clothing and ropes to medical materials
and even environmental technologies like fog collectors. The main obstacle has always been
the same, producing enough silk.

Farming spiders is not a practical solution. Spiders produce several different types of silk, and
there is no simple way to control which type they make. Many spider species are also
cannibalistic, meaning they may eat each other when kept together. These factors make largescale spider farming extremely difficult.

Instead, scientists can use genetic engineering. By inserting a modified silk gene from a
spider into a bacterium, the bacterium can be turned into a tiny silk factory. In this project,
four different versions of a silk protein used by spiders to protect their eggs were tested.

The results showed that the bacteria were able to produce the silk protein. However, the
amount produced was very low. This makes it difficult to determine whether later purification
problems were caused by low production or by technical challenges in the process.

Although large-scale production is not yet achieved, this project shows that it is possible.
With further research and optimization, bacterial production of spider silk may one day allow
us to use this remarkable material in everyday products even perhaps in clothing made
entirely from spider silk.

• author: Östsjö, Jonas
• supervisor: Cedric Dicko; Juanita Francis
• organization: Pure and Applied Biochemistry; Computational Chemistry
• course: KBKM05 20252
• year: 2026
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Recombinant protein, E.Coli, Silk, IMAC, Applied biochemistry
• language: English
• id: 9223172
• date added to LUP: 2026-03-02 11:09:37
• date last changed: 2026-03-02 11:09:37