🌟 Fun & Educational Summary of “Functional expression of recombinant insulins in Saccharomyces cerevisiae”

Imagine you want to mass-produce insulin from different animals (human, cow, pig, chicken) — but using yeast instead of pancreas. That’s exactly what this study accomplishes. The authors redesigned insulin so yeast can secrete it more efficiently, fold it properly, and allow simple purification. Then they demonstrated that the resulting insulin works!

Below is a breakdown of every key concept, method, and result.

🧬 Why This Study Matters

• The global need for insulin is increasing due to rising diabetes rates.
• Cultured meat production also needs animal-specific insulin to replace fetal bovine serum (FBS) in growth media.
• Producing insulin cheaply and efficiently is essential — and yeast could be the perfect “factory.”

But… expressing insulin in yeast is hard. Insulin normally forms inside pancreatic β-cell granules, not in a microbial secretion system.

This study engineers a full pipeline to solve all of those problems.

🧩 The Big Idea of the Paper

The authors propose a new method:

Replace the C-peptide of insulin with a special hydrophilic fusion partner (HL18)

→ boosts secretion and adds a purification tag.

Use yeast’s Kex2 protease to process the precursor into mature insulin

→ avoids unwanted cleavage problems caused by trypsin.

Strengthen yeast's folding machinery (UPR activation) by overexpressing HAC1

→ helps insulin form correct disulfide bonds.

Produce not just human insulin but also bovine, porcine, and chicken insulin

→ important for cultured meat and biomedical applications.

🍰 Breakdown of All Key Topics & Methods

Below is a topic-by-topic guide with explanations of what, how, why, and outcome.

1️⃣ Insulin Structure Refresher

• Insulin is normally made as proinsulin with A-chain + B-chain connected by a C-peptide.
• The C-peptide is later removed by proteolysis to form mature insulin.

Why this matters here

The C-peptide isn't required for function — so the authors replace it with a functional module (HL18) to improve secretion.

📍The multiple sequence alignment figure on page 4 shows that while A/B chains are conserved across species, the C-peptide is not. This justifies replacing it.

2️⃣ HL18 Fusion Partner — why it’s clever

HL18 is a hydrophilic 18-amino-acid peptide derived from yeast VOA1.

Purpose

• Enhances secretion efficiency.
• Carries a His-tag → allows purification using IMAC.
• Replaces the C-peptide.

Outcome

The strain expressing HL18-bINS produces the highest amount of insulin precursor (see SDS-PAGE on page 4).

3️⃣ Expression Constructs

The authors design 3 versions of bovine insulin:

1. bINS – full proinsulin with C-peptide
2. bINSΔC – proinsulin without C-peptide
3. HL18-bINS – C-peptide replaced by HL18 fusion partner

Each construct is placed under:

• GAL10 promoter
• MFα secretion leader
• URA3 marker
• Insulin coding sequence (Fig. 2 shows full plasmid design.)

Why use MFα secretion signal?

It routes the protein through the yeast secretory pathway.

Why GAL10 promoter?

Strong expression even without induction in their strain background.

4️⃣ SDS-PAGE to Evaluate Expression

Method:

• Culture yeast → collect supernatant → precipitate with acetone → run SDS-PAGE with and without β-mercaptoethanol.

Why this method?

• Checks whether insulin precursors are secreted.
• Checks disulfide bond formation (nonreducing vs reducing gel).

Outcome

• HL18-bINS shows strongest bands, meaning highest secretion.
• Under nonreducing conditions, bands smear upward → disulfide-bonded chains (insulin-like structure). (Page 4, Fig. 2C.)

5️⃣ Why Not Use Trypsin?

Trypsin cleaves at Lys/Arg. But the B-chain of insulin contains internal Lys/Arg sites, so trypsin produces unwanted fragments → broken insulin.

Instead, the authors use…

6️⃣ Kex2 Protease — the key innovation

Kex2p cleaves specifically after dibasic residues Arg-Arg or Lys-Arg.

Why use Kex2p?

• Insulin’s B-chain naturally ends in Arg-Arg → correct target for Kex2p.
• No unwanted internal cleavage → better yield of correct insulin.

How they controlled cleavage

The construct includes three Kex2p sites:

1. After MFα leader → needed in vivo
2. Between B-chain and HL18-A-chain → needed in vivo
3. Before HL18 tag → should NOT be cleaved in vivo (so the tag stays attached until purification)

To control the 3rd site, they altered amino acids to give it a low ProP score (0.228) so Kex2p wouldn’t recognize it. (Page 5–6.)

Outcome

• Proper folding occurs in yeast (sites 1 and 2).
• HL18 is removed in vitro after purification by adding recombinant Kex2p.

7️⃣ IMAC (Ni-NTA purification)

Because HL18 contains a His-tag, fusion proteins bind Ni-resin.

Workflow (Fig. 3B, page 5–6)

1. 1st IMAC → purify HL18-insulin precursor
2. Add Kex2p in vitro → HL18 is cleaved off
3. 2nd IMAC → His-tagged HL18 binds column; Mature insulin flows through

Outcome

This produces pure insulin with proper A/B chain disulfide bonding.

8️⃣ Fed-Batch Fermentation

Method:

• 5-L fermenter
• Controlled aeration, pH, and glucose feed
• Samples collected every 4 h

Why use fed-batch?

• Yeast grows to higher density → more secreted insulin.
• Maintains glucose levels for constant productivity.

Outcome

~120–140 mg fusion protein → ~16–25 mg final insulin after purification (Table 1).

9️⃣ UPR Enhancement via HAC1 Overexpression

HAC1 activates the unfolded protein response, increasing:

• Chaperone levels
• ER folding capacity
• Ability to form disulfide bonds

Why needed?

Insulin folding requires three disulfide bonds → difficult in yeast.

Outcome

• HAC1 strain produces insulin with higher biological activity.
• SDS-PAGE confirms proper A/B chain processing.
• MTT assay shows improved cell-growth stimulation vs WT strain. (Fig. 4, page 7.)

🔟 Biological Activity Assay (MTT on HaCaT cells)

Method:

• Add purified insulin at various concentrations to keratinocyte cells.
• Insulin stimulates growth → converted MTT dye indicates viability.

Why this method?

• Simple, quantitative way to test whether recombinant insulin activates insulin signaling.

Result

Insulin from all species (human, cow, pig, chicken) showed normal biological activity, comparable to pancreatic insulin. (Fig. 5B.)

1️⃣1️⃣ Production of hINS, pINS, cINS

After success with bovine insulin, authors used site-directed mutagenesis to swap sequences to human, pig, chicken.

Why easy to implement?

A- and B-chains are highly conserved across species → only minor sequence changes required.

Outcome

All versions:

• Were produced in yeast
• Folded properly
• Purified via same workflow
• Retained biological activity (Shown in Fig. 5.)

🎯 Final Takeaways

✨ Main Achievements

• Built a universal yeast platform for efficient production of multiple insulin types.
• Boosted secretion by HL18 fusion partner.
• Ensured correct processing via controlled Kex2 cleavage.
• Improved folding via HAC1 overexpression.
• Verified full biological activity using cell proliferation assays.

🌱 Why this matters for cultured meat

Different animal cells require species-matched insulin. Producing bovine, pig, and chicken insulin affordably could dramatically reduce the cost of serum-free media in cultured meat production.

🧪 Why this matters for biotech

A blueprint for expressing disulfide-rich peptides in yeast — applicable to hormones, growth factors, enzymes.