Expression and Purification of Human Stem Cell Factor and Its Effect on the Growth of A549 Cells

IF 1.4 4区生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY

The Protein Journal Pub Date : 2025-06-02 DOI:10.1007/s10930-025-10273-w

Zhenling Ma, Xulu Han, Yiyang Zhu, Panpan Fu, Zhenzhen Li, Yongqin Zhao, Junzheng Yang, Wei Liu

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引用次数: 0

Abstract

Stem cell factor (SCF) is a type of hematopoietic cytokine that functions in early hematopoietic stem cells. SCF, together with its cognate receptor c-kit, plays crucial roles in cell survival, proliferation, and differentiation. A dysregulated SCF/c-kit system is reportedly, associated strongly with various kinds of cancer in humans. However, the function and mechanism of SCF in non-small cell lung cancer (NSCLC) remains to be elucidated to date. In this context, the present study attempted to purify two different sizes of human SCF proteins and then investigate the effect of these exogenous SCFs in A549 cells. First, the homo sapiens Kit ligand (KITG) transcript variants a and b were amplified, and recombinant pET-30a vectors were constructed. Soluble His-SCF and His-SCF-2 were then expressed and purified using 0.1 mM isopropyl-β-D-1-tiogalactopiranoside (IPTG) at 25 °C and 0.3 mM IPTG at 25 °C, respectively. MTT assay was conducted next, which revealed that the purified proteins significantly promoted A549 cell activity and increased the phosphorylation of P65. Further, NF-κB pathway blockade was achieved through treatment with BAY11-7082, and it was observed to decrease the A549 cell activity promoted by the purified proteins. These findings provided insights into the biological role of SCF in the development of NSCLC.

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人干细胞因子的表达纯化及其对A549细胞生长的影响。

干细胞因子（Stem cell factor， SCF）是一种造血细胞因子，在早期造血干细胞中起作用。SCF及其同源受体c-kit在细胞存活、增殖和分化中起着至关重要的作用。据报道，失调的SCF/c-kit系统与人类各种癌症密切相关。然而，SCF在非小细胞肺癌（NSCLC）中的作用和机制尚不清楚。在此背景下，本研究试图纯化两种不同大小的人SCF蛋白，然后研究这些外源SCF在A549细胞中的作用。首先，扩增智人Kit配体（Kit ligand, Kit）转录物变体a和b，构建重组pET-30a载体。可溶性His-SCF和His-SCF-2分别用0.1 mM异丙基-β- d -1-半乳糖苷（IPTG）在25°C和0.3 mM IPTG在25°C下表达和纯化。MTT实验显示纯化后的蛋白显著促进A549细胞活性，增加P65的磷酸化。此外，通过BAY11-7082处理可阻断NF-κB通路，并观察到纯化蛋白可降低A549细胞活性。这些发现为探究SCF在非小细胞肺癌发展中的生物学作用提供了新的思路。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

The Protein Journal 生物-生化与分子生物学

CiteScore

5.20

自引率

0.00%

发文量

审稿时长

12 months

期刊介绍： The Protein Journal (formerly the Journal of Protein Chemistry) publishes original research work on all aspects of proteins and peptides. These include studies concerned with covalent or three-dimensional structure determination (X-ray, NMR, cryoEM, EPR/ESR, optical methods, etc.), computational aspects of protein structure and function, protein folding and misfolding, assembly, genetics, evolution, proteomics, molecular biology, protein engineering, protein nanotechnology, protein purification and analysis and peptide synthesis, as well as the elucidation and interpretation of the molecular bases of biological activities of proteins and peptides. We accept original research papers, reviews, mini-reviews, hypotheses, opinion papers, and letters to the editor.