Soluble expression and functional characterization of multi-subunit hemoglobins from the blood clam, Tegillarca granosa

Weifeng Zhang; Tiancheng Chen; Yazhuo Hu; Xuejia Li; Xizhi Shi; Yongbo Bao

doi:10.1016/j.cbpb.2025.111125

Soluble expression and functional characterization of multi-subunit hemoglobins from the blood clam, Tegillarca granosa

Comp Biochem Physiol B Biochem Mol Biol. 2025 Jul 9:279:111125. doi: 10.1016/j.cbpb.2025.111125. Online ahead of print.

Authors

Weifeng Zhang¹, Tiancheng Chen², Yazhuo Hu³, Xuejia Li³, Xizhi Shi⁴, Yongbo Bao⁵

Affiliations

¹ School of Marine Science, Ningbo University, Ningbo 315832, China; Key Laboratory of Aquatic Germplasm Resource of Zhejiang, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China; Ninghai Institute of Mariculture Breeding and Seed Industry, Zhejiang Wanli University, Ningbo 315604, China.
² Key Laboratory of Aquatic Germplasm Resource of Zhejiang, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China; Ninghai Institute of Mariculture Breeding and Seed Industry, Zhejiang Wanli University, Ningbo 315604, China; College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
³ Key Laboratory of Aquatic Germplasm Resource of Zhejiang, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China; Ninghai Institute of Mariculture Breeding and Seed Industry, Zhejiang Wanli University, Ningbo 315604, China.
⁴ School of Marine Science, Ningbo University, Ningbo 315832, China.
⁵ Key Laboratory of Aquatic Germplasm Resource of Zhejiang, College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, China; Ninghai Institute of Mariculture Breeding and Seed Industry, Zhejiang Wanli University, Ningbo 315604, China. Electronic address: baoyongbo@zwu.edu.cn.

PMID: 40645559
DOI: 10.1016/j.cbpb.2025.111125

Abstract

The Escherichia coli expression system, combined with site-directed mutagenesis, is a powerful tool for investigating the structure-function relationship of hemoglobin (Hb). However, producing biologically active recombinant hemoglobin (rHb) from invertebrates, especially in multimeric forms, remains difficult. Here, we describe the recombinant expression, purification, and characterization of homodimeric rHbI and heterotetrameric rHbII from the blood clam Tegillarca granosa. Optimization of expression conditions demonstrated that high yields (65.4-135.6 g/L) of soluble T. granosa rHbs could be achieved using the JM109(DE3) strain with 15 °C induction, 0.25 mM isopropyl β-D-thiogalactoside (IPTG) levels, and codon optimization. Circular dichroism and ultraviolet-visible spectroscopy verified that purified rHbs exhibited a secondary structure similar to that of native Hbs (nHbs) and preserved their diverse ligand-binding capacities. Oxygen-binding assays further showed that the oxygen affinity of rHbs was comparable to that of nHbs. Nitrite reduction assays revealed that T. granosa nHbs exhibited weak nitrite reductase activity (0.049-0.116 M^-1 s^-1 at 25 °C), generally lower than that of vertebrate globins. However, rHbs, particularly rHbI, displayed substantially higher reduction rate constants (0.147-4.589 M^-1 s^-1 at 25 °C) than nHbs, possibly due to differences in structural conformation or the N-terminal tag carried by rHbs. Overall, the recombinant expression and purification method established in this study provides a valuable framework for future research on the structure-function relationships of T. granosa Hb and invertebrate Hbs more broadly.

Keywords: Escherichia coli; Function; Hemoglobin; Recombinant expression; Tegillarca granosa.