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Lowry method for quantitative detection of protein concentration

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The determination of protein content is one of the important indicators for the quality control of biotechnology drugs. Lowry method is a classic method for quantitative determination of protein content, which is widely used and suitable for identifying the purification effect of protein and carrying out activity determination. Lowry method and BCA method can be used to determine the content of protein impurities in purified polysaccharides. Protein separation and purification techniques are widely used in biochemistry research because in order to study a protein, it is necessary to first isolate and purify the protein from other protein molecules.

In the field of biotech drugs research and development, the accuracy of the determination of protein content in the product specifications and packaging have guiding significance, is better than active calculation, control and other physical and chemical properties of set limit to determine residual impurities, the basis of preclinical safety and efficacy evaluation research in the effective dose and toxic dose setting as well as the important basis for formulating clinical scheme.The principle of Lowry method for the determination of protein content is that protein peptide bond and Cu2+ chelate in alkaline solution, forming protein-copper complex, reducing phenylphosphomolybdic acid, producing blue compound, blue depth and protein concentration is linear relationship, under certain conditions, blue depth is proportional to the amount of protein.

The advantage of Lowry method for the determination of protein content is that it is more commonly used, sensitive and more sensitive than biuret method.The disadvantage is that it has poor specificity, many interfering substances (such as TRIS buffer, sucrose, ammonium sulfate, sulfhydrates, phenols and citric acid, etc.), the linear relationship of the standard curve is not particularly strict.A large number of purified recombinant proteins can be used for drug screening, structural biology research, cell biology research, proteomics and a series of biomedical research.Medisi has successfully built a complete protein expression technology platform for Escherichia coli prokaryotic cells and yeast eukaryotic cells, and can provide a complete set of services including protein expression plasmid construction, protein expression condition exploration and protein purification according to customer requirements.

Heme oxygenase (HO) is one of the important components of microsomal enzyme system. It can catalyze the degradation of heme to produce bilirubin, free iron and carbon monoxide.HOs have three isoenzymes :HO-1, HO-2, and HO-3.HO-2 is mainly distributed in brain, retina, nervous system and other tissues.In order to obtain the purified heme oxygenase-2 with biological activity for research, the expression system of Escherichia coli can be used to express the heme oxygenase-2. Some purification and separation technologies can be used to obtain the purified target protein, and then the purification effect and activity determination can be carried out by Lowry method and other methods.

Some researchers expressed and purified the recombinant heme oxygenase-2 (HO-2) plasmid pMW172a/HO-2 into Escherichia coli BL-21 to study the effects of culture temperature and shaking speed on the expression of HO-2 protein, and to determine the optimal conditions for soluble expression.The target protein was purified by over-speed centrifugation, fractional salting-out and chromatography.SDS-PAGE, Lowry method, spectral scanning and other methods were used to identify the purification effect and determine the activity [1].Results High purity and active target protein was obtained, which provided a feasible purification route for further study on the relationship between structure and function of HO-2.

Lowry method is widely used in the determination of protein content, and researchers have verified the Lowry method for the determination of recombinant human serum albumin-interleukin-2 fusion protein content, providing a method for the detection of recombinant human serum albumin-interleukin-2 fusion protein content for subsequent experiments [2].The standard curve was established in the concentration range of 100~0μg/ mL and the correlation coefficient (R~2) was greater than 0.99.The accuracy of at least 6 points on the standard curve (including both ends) is between 95.2% and 111.67%, and the accuracy of the lower limit of the standard curve is between 95.98% and 103.6%.The intra-batch precision of quality control samples with high, medium and low mass concentrations (80,40,20μg/ mL) ranged from 1.51% to 4.41%, and the inter-batch precision ranged from 3.83% to 6.36%.Based on the above information, the Lowry method for the determination of recombinant human serum albumin-interleukin-2 fusion protein content is stable and reproducible.

Protein is the material basis of life, is the organic macromolecule, is the basic organic matter that constitutes the cell, is the main undertaker of life activities.Protein content determination is a basic operation in the laboratory, but due to the complexity of protein composition and properties, it is necessary to consider the advantages and disadvantages of various measurement methods in the determination of protein content, and then choose the appropriate measurement method.

[1] Expression and purification of recombinant heme oxygenase-2 in Escherichia coli [1].

[2] Lowry method for the determination of recombinant human serum albumin-interleukin-2 fusion protein content [2].

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