Nisin-inducible secretion of a biologically active single-chain insulin analog by Lactococcus lactis NZ9000

The strategy of translationally fusing the α- and β-subunits of human chorionic gonadotropin (hCG) into a single-chain molecule has been used to produce novel analogs of hCG. Previously we reported expression of a biologically active single-chain analog hCGαβ expressed using Pichia expression system. Using the same expression system, another analog, in which the α-subunit was replaced with the second β-subunit, was expressed (hCGββ) and purified. hCGββ could bind to LH receptor with an affinity three times lower than that of hCG but failed to elicit any response. However, it could inhibit response to the hormone in vitro in a dose-dependent manner. Furthermore, it inhibited response to hCG in vivo indicating the antagonistic nature of the analog. However, it was unable to inhibit human FSH binding or response to human FSH, indicating the specificity of the effect. Characterization of hCGαβ and hCGββ using immunological tools showed alterations in the conformation of some of the epitopes, whereas others were unaltered. Unlike hCG, hCGββ interacts with two LH receptor molecules. These studies demonstrate that the presence of the second β-subunit in the single-chain molecule generated a structure that can be recognized by the receptor. However, due to the absence of α-subunit, the molecule is unable to elicit response. The strategy of fusing two β-subunits of glycoprotein hormones can be used to produce antagonists of these hormones.

Download Full-text

The relationship between the connecting peptide of recombined single chain insulin and its biological function

Science in China Series C Life Sciences ◽

10.1007/bf02879353 ◽

2001 ◽

Vol 44 (6) ◽

pp. 593-600 ◽

Cited By ~ 8

Author(s):

Yiding Huang ◽

Zhenhe Liang ◽

Youmin Feng

Keyword(s):

Biological Function ◽

Single Chain ◽

Single Chain Insulin ◽

The Relationship

Download Full-text

Intracellular and Extracellular Expression of Bacillus thuringiensis Crystal Protein Cry5B in Lactococcus lactis for Use as an Anthelminthic

Applied and Environmental Microbiology ◽

10.1128/aem.02365-15 ◽

2015 ◽

Vol 82 (4) ◽

pp. 1286-1294 ◽

Cited By ~ 8

Author(s):

Evelyn Durmaz ◽

Yan Hu ◽

Raffi V. Aroian ◽

Todd R. Klaenhammer

Keyword(s):

Bacillus Thuringiensis ◽

Lactococcus Lactis ◽

Copy Number ◽

Oral Delivery ◽

Membrane Integrity ◽

Biologically Active ◽

High Copy Number ◽

Cry Protein ◽

Western Blots ◽

Content Type

ABSTRACTTheBacillus thuringiensiscrystal (Cry) protein Cry5B (140 kDa) and a truncated version of the protein, tCry5B (79 kDa), are lethal to nematodes. Genes encoding the two proteins were separately cloned into a high-copy-number vector with a strong constitutive promoter (pTRK593) inLactococcus lactisfor potential oral delivery against parasitic nematode infections. Western blots using a Cry5B-specific antibody revealed that constitutively expressed Cry5B and tCry5B were present in both cells and supernatants. To increase production,cry5Bwas cloned into the high-copy-number plasmid pMSP3535H3, carrying a nisin-inducible promoter. Immunoblotting revealed that 3 h after nisin induction, intracellular Cry5B was strongly induced at 200 ng/ml nisin, without adversely affecting cell viability or cell membrane integrity. Both Cry5B genes were also cloned into plasmid pTRK1061, carrying a promoter and encoding a transcriptional activator that invoke low-level expression of prophage holin and lysin genes inLactococcuslysogens, resulting in a leaky phenotype. Cry5B and tCry5B were actively expressed in the lysogenic strainL. lactisKP1 and released into cell supernatants without affecting culture growth. Lactate dehydrogenase (LDH) assays indicated that Cry5B, but not LDH, leaked from the bacteria. Lastly, using intracellular lysates fromL. lactiscultures expressing both Cry5B and tCry5B,in vivochallenges ofCaenorhabditis elegansworms demonstrated that the Cry proteins were biologically active. Taken together, these results indicate that active Cry5B proteins can be expressed intracellularly in and released extracellularly fromL. lactis, showing potential for future use as an anthelminthic that could be delivered orally in a food-grade microbe.

Download Full-text

Age of diabetes onset in the mutant proinsulin syndrome correlates with mutational impairment of protein foldability and stability

10.1101/2021.09.24.461687 ◽

2021 ◽

Author(s):

Balamurugan Dhayalan ◽

Yen-Shan Chen ◽

Yanwu Yang ◽

Mark Jarosinski ◽

Deepak Chatterjee ◽

...

Keyword(s):

Molten Globule ◽

Disease Onset ◽

Single Chain ◽

Human Insulin Gene ◽

Disulfide Pairing ◽

Single Chain Insulin ◽

Hormone Analogs ◽

Α Helix ◽

Core Packing

Diverse heterozygous mutations in the human insulin gene cause a monogenic diabetes mellitus (DM) syndrome due to toxic misfolding of the variant proinsulin. Whereas mutations that add or remove cysteines (thereby leading to an odd number of thiol groups) generally lead to neonatal-onset DM, non-Cys-related mutations can be associated with a broad range of ages of onset. Here, we compare two mutations at a conserved position in the central B-chain α-helix: one neonatal in DM onset (ValB18→Gly) and the other with onset delayed until adolescence (AlaB18). The substitutions were introduced within a 49-residue single-chain insulin precursor optimized for folding efficiency (Zaykov, A., et al. ACS Chem. Biol. 9, 683-91 (2014)). Although mutations are each unfavorable, GlyB18 (a) more markedly perturbs DesDi folding efficiency in vitro than does AlaB18 and (b) more severely induces endoplasmic reticulum (ER) stress in cell-based studies of the respective proinsulin variants. In corresponding two-chain hormone analogs, GlyB18 more markedly perturbs structure, function and thermodynamic stability than does AlaB18. Indeed, the GlyB18-insulin analog forms a molten globule with attenuated α-helix content whereas the AlaA18 analog retains a nativelike cooperative structure with reduced free energy of unfolding (ΔΔGu 1.2(±0.2) kcal/mole relative to ValB18 parent). We propose that mutations at B18 variably impede nascent pairing of CysB19 and CysA20 to an extent correlated with perturbed core packing once native disulfide pairing is achieved. Differences in age of disease onset (neonatal or adolescent) reflect relative biophysical perturbations (severe or mild) of an obligatory on-pathway protein folding intermediate.

Download Full-text