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High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

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Characterizing and Improving pET Vectors for Cell-free Expression.

Kara Jew¹, Philip E J Smith², Byungcheol So²

¹Biological Sciences Department, California Polytechnic State University, San Luis Obispo, CA, United States.

Frontiers in Bioengineering and Biotechnology

|July 11, 2022

Summary

Optimizing cell-free protein synthesis (CFPS) involves modifying plasmid DNA. Removing specific elements like the lac operator from pET vectors significantly enhances protein production in various CFPS systems.

Keywords:

cell-free in vitro pET30 protein synthesis template translation

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Area of Science:

Biotechnology
Molecular Biology
Synthetic Biology

Background:

Cell-free protein synthesis (CFPS) offers versatile applications but is limited by plasmid vector performance.
Commonly used bacterial expression vectors, like the pET series, often yield suboptimal protein production in CFPS.

Purpose of the Study:

To investigate the impact of expression cassette elements within the pET30 vector on protein yield in CFPS.
To identify specific DNA sequences responsible for reduced protein production in CFPS systems.

Main Methods:

Systematic deletion of genetic elements from the pET30 vector backbone.
Evaluation of protein production using three distinct CFPS systems: NEBExpress, PURExpress, and CFAI-based E. coli extracts.

Main Results:

Identified the lac operator (lacO) and N-terminal tags as key elements hindering protein production in pET vectors.
Demonstrated that removing these elements from pET vectors significantly enhances protein yields in extract-based CFPS.

Conclusions:

Vector backbone sequence elements critically influence protein production efficiency in CFPS.
Modified pET vectors, lacking lacO and specific tags, support high-titer protein expression in diverse CFPS platforms, improving accessibility for researchers.